RSV-F proteins

Abstract

The present disclosure provides, inter alia, a respiratory syncytial virus fusion (RSV-F) protein in a pre-fusion conformation that is mutated relative to a wild-type RSV-F according to SEQ ID NO: 1; wherein the RSV-F protein comprises at least one mutation relative to the wild type in the region corresponding to positions 217-239 of SEQ ID NO: 1; wherein the at least one mutation is introduced by substitution or insertion into a residue comprising a hydrogen bond donor and / or acceptor moiety in the side chain.

Description

Technical Field

[0001] The present disclosure is in the field of vaccinology, particularly structure-based protein design of vaccine antigens. Background Art

[0002] Respiratory syncytial virus (“RSV”) is a ribonucleic acid virus of the family Pneumoviridae, which exists in two antigenically distinct subgroups, called RSV A and RSV B.

[0003] RSV is a major cause of hospitalization and death in infants and the elderly. RSV causes approximately 58,000 hospitalizations and 100 - 500 deaths in children under five years old in the United States each year [1], while 177,000 hospitalizations and 14,000 deaths in adults 65 years old and older [2]. Therefore, developing a safe and effective vaccine to prevent severe diseases and hospitalizations caused by RSV is a high priority.

[0004] The antiviral drug ribavirin is the only approved antiviral therapy for RSV treatment, but in infants and young children, its use is limited to severe hospitalization cases [3]. In addition, two RSV-specific humanized monoclonal antibodies, palivizumab (Synagis) and motavizumab, have been confirmed to be safe and effective in reducing RSV hospitalization rates and severe complications in high-risk children in multiple clinical settings [4, 5, 6, 7, 8]. RSV treatment available in the elderly is generally supportive, consisting of supplemental oxygen, intravenous fluids, and bronchodilators. In May 2023, the first RSV vaccine was approved by the FDA (AREXVY, for the elderly). However, there is clearly still a need for other safe and effective RSV prophylactic vaccines.

[0005] Structure-based antigen design may be the key to developing such a vaccine. The RSV fusion protein (“RSV-F”) in the viral envelope is the most effective target for neutralizing antibodies such as motavizumab. Recent advances in the structural biology of RSV-F have revealed changes in its antigenic characteristics that occur during the fusion process between the viral envelope and the host cell membrane. RSV-F adopts a metastable “pre-fusion” conformation as a homotrimer in the viral envelope and then an irreversible and unique “post-fusion” conformation during fusion with the host cell membrane (see Figure 2 ) of [9]. This pre-fusion conformation is more immunogenic and is bound by most RSV-F-specific neutralizing antibodies in human serum. However, the native pre-fusion conformation is not energetically favorable. Therefore, the pre-fusion RSV-F antigen for vaccine use needs to be stabilized to prevent irreversible folding into the post-fusion conformation.

[0006] Structure-based antigen design strategies have previously been used in attempts to stabilize the pre-fusion conformation. However, there remains a need for a pre-fusion RSV-F protein design that can be used as a vaccine antigen and, in particular, that is amenable to high expression yields when expressed from nucleic acid. SUMMARY OF THE INVENTION

[0007] The inventors have generated novel RSV-F proteins in the pre-fusion conformation.

[0008] Using a computational model of wild-type pre-fusion RSV-F (strain A2), the inventors first identified an in silico residue substitution landscape that enhances the expression and stability of trimeric pre-fusion RSV-F (see, e.g., Example 2). This strategy utilizes a combination of sequence-based evolutionary bioinformatics and structure-based thermodynamic design. Successive rounds of expression, characterization, and validation of the pre-fusion structure then narrowed this landscape to identify a smaller set of substitutions capable of achieving the pre-fusion conformation (see, e.g., Examples 3-5). In vitro screening then revealed a single substitution that drives this pre-fusion conformation (see, e.g., Example 6).

[0009] The introduction of disulfide bonds and / or proline (P) residues is a common stabilization strategy in structure-based antigen design and has previously been applied to RSV-F (see, e.g., [9,10]). However, in vitro screening by the inventors surprisingly revealed that a single substitution (without introducing either of the above) is capable of generating pre-fusion RSV-F (see, e.g., Example 6, Design F310, which has an N228K substitution). Three-dimensional structure analysis by the inventors revealed that the introduced residue (K) forms an intra-protomer hydrogen bond (H-bond) with the proximal residue Y250 (see Figure 25 ). To the inventors' knowledge, the achievement of the pre-fusion conformation of RSV-F by a single substitution relative to wild-type (and furthermore, which results in an intra-protomer H-bond) has not been reported. Thus, this type of mutation (preferably a substitution) can be used alone to achieve pre-fusion RSV-F per se, or in combination with further mutations (preferably substitutions) to stabilize RSV-F in the pre-fusion conformation (e.g., to provide longer-term stability). In addition, exemplary RSV-F proteins according to the present disclosure exhibit higher expression yields in vitro than DS-Cav1 of reference

[10] (see, e.g., Examples 4 and 6; Figure 8 and Figure 18 ). Exemplary RSV-F proteins according to the present disclosure also exhibit greater long-term stability than DS-Cav1 (see, e.g., Example 9; Figure 32).

[0010] In the in vivo context, when administered in a murine model, an exemplary RSV-F protein according to the present disclosure elicits a pre-fusion RSV-F specific antibody response and also elicits a neutralizing antibody response against, for example, RSV A (see, for example, Example 10, Example 11 and Example 13; Figures 34 - 37, Figure 43 and Figure 44).

[0011] In view of the above, the RSV-F protein generated by the present inventors can be used as a vaccine antigen, i.e., for prophylactic vaccination against RSV.

[0012] Accordingly, in a first independent aspect, the present disclosure provides:

[0013] An RSV-F protein in a pre-fusion conformation, which is mutated relative to the wild-type RSV-F according to SEQ ID NO:1; wherein the RSV-F protein comprises at least one mutation relative to the wild-type in the region corresponding to positions 217 - 239 of SEQ ID NO:1; wherein the at least one mutation is introduced by substitution or insertion of a residue containing an H-bond donor and / or acceptor moiety in the side chain.

[0014] In a further independent aspect, the present disclosure provides a nucleic acid (preferably RNA) encoding the RSV-F protein of the present disclosure.

[0015] In a further independent aspect, the present disclosure provides a host cell comprising the nucleic acid of the present disclosure.

[0016] In a further independent aspect, the present disclosure provides an in vitro method for producing the RSV-F protein of the present disclosure, which comprises expressing the nucleic acid (preferably, an expression vector) of the present disclosure in a host cell, and optionally purifying the RSV-F protein.

[0017] In a further independent aspect, the present disclosure provides a vector (preferably, a lipid nanoparticle) comprising the nucleic acid of the present disclosure.

[0018] In a further independent aspect, the present disclosure provides a pharmaceutical composition comprising the RSV-F protein, nucleic acid (preferably RNA) or vector (preferably lipid nanoparticle) of the present disclosure.

[0019] In a further independent aspect, the present disclosure provides the RSV-F protein, nucleic acid (preferably RNA), vector (preferably lipid nanoparticle) or pharmaceutical composition of the present disclosure for medical use.

[0020] In a further independent aspect, the present disclosure provides a method of treatment comprising the step of administering to a subject (preferably a subject in need thereof) an effective amount of an RSV-F protein, nucleic acid (preferably RNA), vector (preferably a lipid nanoparticle), or pharmaceutical composition of the present disclosure.

[0021] Further independent aspects of the present disclosure are provided throughout the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1. Consensus design based on sequence and structure to stabilize the pre-fusion conformation of RSV-F. (A) The ROSETTA protein design suite was used to find combinatorial substitutions at different computational simulation energy thresholds, generating 12 sequences, ranging from -0.5 kcal / mol to -6 kcal / mol (in 0.5 kcal / mol increments) relative to the wild type; (B) The subsequent substitution panorama is shaded to illustrate the sequence diversity in the potential design relative to the known epitope positions (sites I, II, III, IV, V) (darker shading represents greater sequence diversity).

[0023] Figure 2 . Expression of the "first round" design relative to DS-Cav1 (from reference

[10] ). Biolayer interferometry (BLI) of the histidine tag sequence showed that designs F21 and F28 (referred to as 21 and 28) were expressed in mammalian cells when compared to spent media (confirmed by subsequent experiments, data not shown).

[0024] Figure 3 . Binding affinity (K D ) of pre-fusion RSV-F-specific antibodies to "first round" RSV-F mutants. The pre-fusion conformations of two designs, F21 and F28, were tested by biolayer interferometry (BLI) against the antibodies AM14 (quaternary epitope), D25 (site ), RSB1 (site V), and motavizumab (site II).

[0025] FIG. 4. Models of the binding of (A) AM14 (quaternary epitope), (B) D25 (site ), (C) motavizumab (site II), and (D) RSB1 (site V) Fabs to wild-type RSV-F. For clarity, only a single copy of the Fab binding to each pre-F trimer is shown.

[0026] Figure 5. Three-dimensional structure in the "first round" design F21 and substitutions relative to the wild type (a trimer is shown, with individual protomers highlighted in dark gray, and substitutions relative to the wild type are shown as spheres).

[0027] Figure 6. Design of "second round" constructs, using the substitution panorama from the "first round" for human RSV-F A2 subtype sequences for (A) all residue designs (including solvent-accessible) or (B) buried only designs. The localization of mutations included in each design scenario is shown in dark coloring.

[0028] Figure 7 . Octet BLI of 15 "second round" sequences (F212 - F226) that bind to RSV-F antibodies (AM14, D25, RSB1, motavizumab), or for which expression was quantified using a histidine tag (the "F" prefix is not used in the figure). Reactions were normalized to DS-Cav1.

[0029] Figure 8 . Quantification of protein yields from 80 ml cultures for 5 "second round" consensus designs compared to DS-Cav1 (the "F" prefix is not used in the figure).

[0030] Figure 9 . Substitutions relative to WT in 5 designs from the "second round".

[0031] Figure 10 . Overview of expression, thermal stability, binding affinity to RSV-F mAb, and antigenicity of the "second round" RSV-F antigen. The second column provides protein expression yields from 80 ml cultures.

[0032] Figure 11 . Three-dimensional structure in the "second round" design F224 and substitutions relative to the wild type. F225 has the same set of substitutions except for A241N (position 241 is A as in the wild type).

[0033] Figure 12 . Three-dimensional structure in the "second round" design F216 and substitutions relative to the wild type.

[0034] Figure 13. (A) Classes of 2D cryo-EM observations of design F21 ("first round", 31 substitutions). (B) cryo-EM density map of design F21 bound to AM14 Fab.

[0035] Figure 14 . cryo-EM density map.

[0036] Figure 15 . cryo-EM density map of engineered F224 (“round 2”, 8 substitutions) in complex with AM14 Fab. cryo-EM density map.

[0037] Figure 16 . cryo-EM parameters for 3D structural analysis of F21, F216, F224, and F310.

[0038] Figure 17 . Round 3 minimal substitution screen - study design. A single reverse substitution was made in the F225 sequence (7 mutations from WT, fewest mutations in successful round 2 constructs) such that each sample would have 6 mutations relative to the WT sequence (left column). Additionally, single substitutions from the F225 sequence were added to WT individually (right column).

[0039] Figure 18 . (A) Protein yields of round 3 minimal substitution designs relative to DS-Cav1. Also shown are negative controls (EXPIFECTAMINE and cell culture supernatant), F225, and F300 (wild type). F225 showed comparable yields in round 3 (white bars) and round 2 (dark bars).

[0040] Figure 19 . Octet BLI of “round 3” minimal substitution designs in complex with RSV-F antibodies (AM14, D25, RSB1, motavizumab) relative to DS-Cav1. Also shown are negative controls (EXPIFECTAMINE and cell culture supernatant), F225, and F300 (wild type).

[0041] Figure 20 . Study design for round 3 (mRNA)-only epitope rescue experiment.

[0042] Figure 21 . Percentage of positive cells of “round 2” and “round 3” RSV-F designs and controls (DS-Cav1, positive control RSV-F construct, and negative control JW27 (NCBI:txid65840)) expressed from mRNA, detected by RSB1, AM14, motavizumab, and 4D7 antibodies.

[0043] Figure 22 . Magnified view of substitution S55T in the cryo-EM structure of F21. Thr55 is shown as a stick with a transparent surface. Residues that form the hydrophobic pocket and participate in van der Waals contacts with Thr55 are shown as sticks (including the hydrophobic pocket).

[0044] Figure 23 .Magnified view of the S215A substitution from the F21 cryo-EM structure (including the proximal α-helix). A215 is depicted as a stick with a transparent surface. Residues that form a hydrophobic region and may participate in van der Waals contacts with A215 are shown as sticks.

[0045] Figure 24. (A) Position of the N348 glycan in designed F216; (A)(1) Image of the designed F216 trimer highlighting the position of the N348 glycan (shown as a sphere). The magnified view shows the position of the N348 glycan and the proximal K419D substitution on the neighboring protomer. (B) Position of the N348 glycan in designed F224; (B)(1) Image of the designed 224 trimer highlighting the position of the N348 glycan (shown as a sphere). The magnified view depicts the position of the N348 glycan in designed F224 and the proximal K419 residue (unsubstituted relative to wild type) on the neighboring protomer.

[0046] Figure 25 .Magnified view of the N228K substitution from the cryo-EM structure of F21. K228 and surrounding residues are depicted as sticks. The hydrogen bond between K228 and Y250 is depicted as a dashed line.

[0047] Figure 26 .Cryo-EM density map of designed F310 (“Round 3”, 1 substitution) bound to AM14 Fab

[0048] Figure 27 .HPLC chromatograms evaluating the monodispersity of F310 or F310_v2 (2x Strep tag removed relative to F310) after purification, incubation overnight at 4 °C, or one freeze / thaw cycle.

[0049] Figure 28 .Protein yields of the Round 3 epitope restoration designs relative to DS-Cav1 after nickel affinity purification from 90 mL of cell harvest medium. Protein yields from Round 2 and Round 3 purifications of DS-Cav1 and Round 2 are shown.

[0050] Figure 29 .Octet BLI of the Round 3 epitope restoration designs binding to RSV-F antibodies (AM14, D25, RSB1, motavizumab) relative to DS-Cav1. Also shown are the negative controls from Round 2 (EXPIFECTAMINE and cell culture supernatant), F216, and F217 (discontinuous lines around the bars, data from Figure 7 )

[0051] ​Figure 30. Binding of (A) DS Cav-1, (B) F216, (C) F217, (D) F318, and (E) F319 to RSV antibodies (AM14 and D25) was determined using BIACORE at 50 °C or 60 °C for 30, 60, or 120 min. Results were reported as the response relative to the control (time 0) sample.

[0052] Figure 31 . After incubation at 4 °C or 25 °C for up to 21 days, no changes in long-term stability were observed as determined by changes in thermal stability as measured by nano-DSF. Curves of the melting temperature (Tm) of unincubated or samples incubated at 4 °C or 21 °C for 21 days were plotted in the histogram.

[0053] Figure 32. Binding of the second-round designs F216, F217, F224 (and DS-Cav1 control) to RSV antibodies (AM14, D25, and RSB1) was evaluated using BIACORE after incubation at 4 °C or 25 °C for 21 days. Results were reported as the binding relative to the unincubated protein.

[0054] Figure 33. (A) Total levels of RSV prefusion protein-specific IgG-binding antibodies from mice immunized with a 3 μg dose were measured using Luminex assays. Anti-RSV prefusion IgG antibodies from immunized mice measured in absorbance units per mL are shown at day 21 and day 35. The geometric mean titer (GMT) and 95% confidence intervals (bars) are represented. The GMT values at day 21 and day 35 are shown below. (B) The geometric mean ratio (GMR) and 90% confidence intervals comparing the constructs to DS-Cav1 were calculated. Data at day 21 (bottom) and day 35 (top) are shown. The raw numbers for GMR, lower limit (LL), and upper limit (UP) are shown on the right. At day 21, F224 was statistically similar to DS-Cav1. At day 35, F216 was statistically similar to DS-Cav1. "PreF design 16" = F216, "PreF design 17" = F217, "PreF design 24" = F224, "PreF design 25" = F225.

[0055] Figure 34. (A) The total levels of RSV prefusion protein-specific IgG-binding antibodies from mice immunized with a 0.3 μg dose were measured using a Luminex assay. Anti-RSV prefusion IgG antibodies from immunized mice measured in absorbance units per mL are shown at days 21 and 35. The geometric mean titer (GMT) and 95% confidence intervals (bars) are represented. The GMT values at days 21 and 35 are shown below. The limit of detection (LOD) is represented by the dashed line. (B) The geometric mean ratio (GMR) and 90% confidence intervals were calculated comparing the constructs to DS-Cav1. Data at day 21 (bottom) and day 35 (top) are shown. The raw numbers for GMR, lower limit (LL), and upper limit (UP) are shown on the right. At day 21, F216 and F217 were statistically similar to DS-Cav1. “PreF design 16” = F216, “PreF design 17” = F217, “PreF design 24” = F224, “PreF design 25” = F225.

[0056] Figure 35. (A) At day 35, the RSV neutralizing antibody titers of mice immunized with 3.0 or 0.3 μg doses were measured using a neutralization assay. Group 1 = saline; Group 2 = DS-CAV1 3 μg; Group 3 = DS-CAV1 0.3 μg; Group 4 = F216 3 μg; Group 5 = F216 0.3 μg; Group 6 = F217 3 μg; Group 7 = F217 0.3 μg; Group 8 = F224 3 μg; Group 9 = F224 0.3 μg; Group 10 = F225 3 μg; Group 11 = F225 0.3 μg. Neutralizing antibody levels are shown as circles. The GMT with 95% confidence intervals (bars) is shown. (B) At day 35, the RSV neutralizing antibody titers of mice immunized with 3.0 or 0.3 μg doses were measured using a neutralization assay. The GMR with 90% confidence intervals was calculated. At the 0.3 μg dose, designs F216, F217, F224, and F225 were statistically similar to DS-Cav1. At the 3 μg dose, design F217 was statistically similar to DS-Cav1. “PreF design 16” = F216, “PreF design 17” = F217, “PreF design 24” = F224, “PreF design 25” = F225.

[0057] Figure 36. RSV pre-F IgG binding antibody geometric mean titers at day 21 (3wp1) and day 35 (2wp2) in animals immunized with (A) 2 μg or (B) 0.2 μg of RNA encoding F(ii) construct, DS-Cav1, F216, F217, F317, or F319. Each point represents an individual animal. (C) Statistical analysis: geometric mean ratio, upper confidence interval (UCI), and lower confidence interval (LCI) of the 2 μg dose results.

[0058] Figure 37. RSV A neutralizing antibody titers (ED60) at day 21 (3wp1) and day 35 (2wp2) in animals immunized with (A) 2 μg or (B) 0.2 μg of RNA encoding F(ii), DS-Cav1, F216, F217, F317, or F319. Each point represents an individual animal. (C) Statistical analysis: GMR, UCI, and LCI of the 2 μg dose results.

[0059] Figure 38. Human primary BJ cells supported surface expression of RSV F protein from candidate mRNAs. Representative images from a 4-day time-course assay are shown. As indicated, individual cell nuclei (denoted as ‘) and cell surface RSV F (denoted as “) variant F318, with 3 amino acids removed from the cytoplasmic tail (CT), were captured by indirect immunofluorescence and imaging (10x objective) in cells fixed at approximately 8 hours (A’ and A”), 24 hours (B’ and B”), 48 hours (C’ and C”), 72 hours (D’ and D”), or 96 hours (E’ and E”) post-transfection and labeled with the primary antibody motavizumab. Population distributions corresponding to the representative images in panels A-E and analysis of transfected and labeled BJ cells at approximately 8 hours (F), 24 hours (G), 48 hours (H), 72 hours (i), and 96 hours (J) post-transfection are shown from high-content imaging (HCi). Population distributions of BJ cells fixed 1 hour post-transfection and processed as described above are shown in each panel (F-J) for reference as a representative of non-specific staining. Population distributions were binned and plotted using cell-specific RSV F mean intensity values from high-content imaging (HCi) and analysis by GraphPad Prism.

[0060] Figure 39. Deletion of the RSV F CT increases cell surface expression of prefusion RSV F trimers. Indirect immunofluorescent labeling with the monoclonal antibody AM14, followed by high content imaging and analysis, was performed over a 4-day time course and quantified to evaluate cell surface expression of the RSV F trimer protein. Primary human BJ cells in 96-well format were transfected with mRNA encoding the RSV F variants F(ii)(A), F318(B), F319(C), or F(i)(D) (solid dots, solid lines) or their respective CT deletion variants CTΔ3 (solid dots, dashed lines), ΔCT20 (open circles, dashed lines), or ΔCT (i.e., full CT deletion – open circles, solid lines). Cell monolayers were fixed at specific time points (hours post transfection), then labeled for RSV F and imaged using a 10x objective. For line graphs, each plotted value represents the mean intensity of Alexa647 signal of cells identified by automated image analysis from 9 imaging fields per well. Each point on the line graph represents the mean (μ) + / - 1 standard deviation (σ) from 3 biological replicates. The area under the curve (AUC) (E) and the standard error of the mean (SEM) are shown for each line graph. The means, AUCs, and variability shown on the line graphs and bar graphs were calculated using GraphPad Prism software.

[0061] Figure 40. Total expression of the RSV F protein is increased for mRNA vaccine candidates with CT deletions. Indirect immunofluorescent labeling with the primary anti-RSV F antibody motavizumab, followed by HCi and analysis, was performed over a 4-day time course and quantified to evaluate cell surface expression of the RSV F protein. Primary human BJ cells in 96-well format were transfected with mRNA encoding the RSV F variants F(ii)(A), F318(B), F319(C), or F(i)(D) (solid dots, solid lines) or their respective CT deletion variants CTΔ3 (solid dots, dashed lines), ΔCT20 (open circles, dashed lines), or ΔCT (open circles, solid lines). Cell monolayers were fixed at specific time points (hours post transfection), then labeled and imaged using a 10x objective. For line graphs, each plotted value represents the mean intensity of Alexa647 signal of cells identified by automated image analysis from 9 imaging fields per well. Each point on the line graph represents the mean (μ) + / - 1 standard deviation (σ) from 3 biological replicates. The area under the curve (AUC) (E) and the standard error of the mean (SEM) are shown for each line graph. The means, AUCs, and variability shown on the line graphs and bar graphs were calculated using GraphPad Prism software.

[0062] Figure 41. In vitro validation of mRNA for in vivo studies. Selected mRNAs encoding RSV F were transfected forward into a primary BJ cell monolayer. The cell monolayer was fixed, and RSV F protein expression was evaluated by indirect immunofluorescence combined with HCi and image analysis. The mRNAs encoded RSV F variants, including the DS-CAV1, F(ii), F(iii), and F(i) proteins or the F318 and F319 protein constructs. Results for the corresponding variants lacking 20 amino acids of the CT (ΔCT20) are also shown. RSV F surface protein expression was quantified 1 day post-transfection by labeling cells with the anti-RSV F antibodies motavizumab (A), D25 (E), or AM14 (I), or 3 days post-transfection (motavizumab (C), D25 (G), or AM14 (K)). Mean cell counts for three imaging wells are shown and correspond to RSV F expression values 1 day post-infection (motavizumab (B), D25 (F), or AM14 (J)) or 3 days post-transfection (motavizumab (D), D25 (H), or AM14 (L)). Each graph depicts the mean (μ) + / - 1 standard deviation (σ) from 3 biological replicates as calculated by GraphPad Prism software.

[0063] Figure 42. The short 5-amino acid CT of the RSV F protein (see Table 8, line 6) maximally enhances RSV F protein expression at the intracellular and cell surface. In vitro transcribed mRNAs encoding F(ii) CT length variants (0, 5, 10, 15, 20, 22 amino acids, and full length) were transfected forward into a primary BJ cell monolayer. The cell monolayer was fixed at time points of 20 hours or 47 hours post-transfection. After immunolabeling of the fixed BJ cells, surface-exposed trimeric RSV F ( Figure 42A ) or pre-fusion RSV F of the whole cell ( Figure 42B ) was quantified by high-content imaging. Trimeric pre-fusion RSV F (identified by AM14) or pre-fusion F (identified by D25) was quantified. Each graph depicts the mean (μ) + / - 1 standard deviation (σ) from 3 biological replicates as calculated by GraphPad Prism software. Background staining represents the median from 6 wells treated concomitantly as experimental non-transfected wells.

[0064] Figure 43. RSV pre-F IgG binding antibody geometric mean titers at day 21 (3wp1) and day 35 (2wp2) in animals immunized with (A) 2 μg or (B) 0.2 μg of constructs encoding F(iii), F(i), F(i)ΔCT20, F(ii), F(ii)ΔCT20, DS-Cav1, F318, F318ΔCT20, F319, or F319ΔCT20 (where each point represents an individual animal). Statistical comparisons of constructs (GMR and 90% CI) are presented in (C)-(G).

[0065] Figure 44. RSV A neutralizing antibody titers (ED60) at day 21 (3wp1) and day 35 (2wp2) in animals immunized with (A) 2 μg or (B) 0.2 μg of constructs encoding F(iii), F(i), F(i)ΔCT20, F(ii), F(ii)ΔCT20, DS-Cav1, F318, F318ΔCT20, F319, or F319ΔCT20 (where each point represents an individual animal). Statistical comparisons of constructs (GMR and 90% CI) are presented in (C)-(E).

[0066] Figure 45. The optimal length of the RSV F CT that supports cell surface expression of pre-fusion trimeric RSV F includes a CT of at least 5 but no longer than 10 amino acids. Cell surface expression of pre-fusion trimeric RSV F was evaluated by indirect immunofluorescent labeling with monoclonal antibody AM14 followed by high content imaging and analysis, quantitated across a 4-day time course. Primary human fibroblasts (BJ) in 96-well format were transfected forward with mRNA encoding the RSV F variant F(ii). In (A), selected CT variants are shown. The parental (F(ii), solid line, solid box) was modified by deletion of the RNA sequences encoding the terminal 15 amino acids (F(ii)CTDΔ15, solid line, solid circles), 16 amino acids (F(ii)CTDΔ16, dashed line, solid circles), 17 amino acids (F(ii)CTDΔ17, dashed line, open circles), 20 amino acids (F(ii)CTDΔ20, solid line, open circles), 21 amino acids (F(ii)CTDΔ21, dashed line, solid boxes), or complete deletion of the CT domain (F(ii)CTDΔ25, solid line, open boxes). In (B), the area under the curve as calculated from the line graph in (A) is shown and is extended to include additional CT deletions, and in (C), the same data as in B are depicted as a line graph. Cell monolayers were fixed at specific time points (hours post-transfection) and then RSV F was labeled and imaged using a 10x objective. For the line graph, each plotted value represents the mean intensity of the Alexa647 signal of cells identified from 9 imaging fields per well by automated image analysis. Each point on the line graph represents the mean (μ) + / - 1 standard deviation (σ) from 3 biological replicates. The area under the curve (AUC) and the standard error of the mean (SEM) are shown in (B). The mean, AUC, and variability shown on the line graph and bar graph were calculated by GraphPad Prism software.

[0067] Figure 46. As in Figure 45, but D25 antibody binding was evaluated.

[0068] Throughout this specification, reference to "CTD" (cytoplasmic tail domain) in Figures 38 - 42, 45, and 46 is equivalent to reference to "CT" (cytoplasmic tail). Thus, "CTDΔ20" is equivalent to "ΔCT20", and so on. Detailed Description

[0069] RSV-F protein in pre-fusion conformation

[0070] As noted above, in a first independent aspect, the present disclosure provides an RSV-F protein in a pre-fusion conformation, which is mutated relative to the wild-type RSV-F according to SEQ ID NO:1; wherein the RSV-F protein comprises at least one mutation relative to the wild-type in the region corresponding to positions 217-239 of SEQ ID NO:1; wherein the at least one mutation is introduced by substitution or insertion of a residue comprising an H-bond donor and / or acceptor moiety in the side chain.

[0071] In a second independent aspect, the present disclosure further provides an RSV-F protein in a pre-fusion conformation, which is mutated relative to the wild-type RSV-F according to SEQ ID NO:1; wherein the RSV-F protein comprises a substitution at position 228 of SEQ ID NO:1 with K or A.

[0072] In a third independent aspect, the present disclosure further provides a multimer comprising a plurality of protomers of an RSV-F protein in a pre-fusion conformation, wherein the protomers of the RSV-F protein comprise at least one mutation relative to the wild-type RSV-F according to SEQ ID NO:1; wherein the at least one mutation is introduced or stabilizes a trans-protomer interaction by substitution or insertion. The trans-protomer interaction can be a hydrogen bond or a tertiary cation-pi-anion interaction between residues of two adjacent protomers of the RSV-F protein. Preferably, the tertiary cation-pi-anion interaction occurs between all of (i), (ii) and (iii); (i) and (ii) are positions 232 and 250 (more preferably E232 and Y250) on a first protomer, respectively, and (iii) is position 235 (more preferably R235) on a second protomer (numbered according to SEQ ID NO:1). The at least one mutation can be introduced by substitution or insertion of at least one residue comprising a hydrogen bond donor and / or acceptor moiety in the side chain (and preferably introduced into the region corresponding to positions 217-239 of SEQ ID NO:1). More preferably, the at least one mutation comprises a substitution at position 228 (N) of SEQ ID NO:1 with K, R, Q or N; or a substitution with K, R or Q (preferably K or R, more preferably K). Preferably, the multimer is a trimer. More preferably, the multimer is a homotrimer comprising three protomers of an RSV-F protein, each RSV-F protein comprising at least one mutation as detailed above in this paragraph.

[0073] In a fourth independent aspect, the present disclosure further provides a multimer comprising a plurality of protomers, wherein at least one protomer comprises or consists of an RSV-F protein according to the first or second independent aspect of the present disclosure.

[0074] To avoid ambiguity, the RSV-F protein according to the first or second independent aspect and the protomer of the RSV-F protein according to the second or third independent aspect are the "RSV-F protein of the present disclosure" as referred to herein. The wild-type RSV-F (A2 subtype) sequences of SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:84, and 107 are not the "RSV-F protein of the present disclosure" as referred to herein. The wild-type RSV-F sequence of SEQ ID NO:108 (B subtype strain 18537) is also not the "RSV-F protein of the present disclosure" as referred to herein.

[0075] As used herein, "mutation" encompasses substitutions, insertions, and deletions of residues, although substitutions and insertions are preferred and substitutions are more preferred according to all aspects of the present disclosure. A mutation that "introduces" a given residue by substitution or insertion may be interchangeably referred to as a "substitution or insertion" of the residue.

[0076] The RSV-F proteins of the present disclosure and the mutations thereof generally included relative to SEQ ID NO:1 are all "engineered". Thus, to the knowledge of the inventors, the RSV-F proteins of the present disclosure are not naturally occurring. In the following sense, the mutations included therein are generally "engineered", i.e., such mutations may occur naturally individually, but have been deliberately selected and introduced into the protein to facilitate the pre-fusion conformation. The RSV-F proteins of the present disclosure can also be considered "recombinant" (in this context, "engineered" and "recombinant" can be used interchangeably). The RSV proteins of the present disclosure generally include engineered mutations relative to SEQ ID NO:1, as defined throughout the present disclosure. SEQ ID NO:1 is the RSV-F sequence of a human RSV strain of subtype A2, which contains two mutations (K66E and Q101P) relative to GenBank accession number KT992094 and is referred to herein as "wild type". To the knowledge of the inventors, the substitutions K66E and Q101P in the F protein were generated by passage of the A2 strain deposited under GenBank accession number KT992094 (also wild type), see, for example,

[11] . Thus, for the purposes of the present disclosure, SEQ ID NO:1 (which contains these two substitutions) is referred to as "wild type" (in accordance with, for example,

[12] ). References to "wild-type RSV-F according to SEQ ID NO:1" and "SEQ ID NO:1" can be used interchangeably herein. SEQ ID NO:1 does not include the trimerization domain, transmembrane domain or cytoplasmic domain at the C-terminus, because when used as a vaccine antigen (e.g., an RSV-F protein-based vaccine or a nucleic acid-based vaccine encoding RSV-F), the domain(s) included at the C-terminus may vary depending on the format of the RSV-F protein. The RSV proteins of the present disclosure may also include mutations relative to SEQ ID NO:1 that are present in RSV-F proteins from other naturally occurring and engineered strains and subtypes (e.g., RSV-F proteins of other subtype A or subtype B strains). Thus, the RSV-F proteins of the present disclosure can be of subtype A or subtype B.

[0077] "wherein the at least one mutation increases the hydrophobicity of the [given sequence / region] relative to the wild-type [corresponding sequence / region]" means that as a result of the at least one mutation, the sum hydrophobicity of all residues in the region is increased relative to the corresponding wild-type region. For example, considering a single substitution in a given sequence / region, S can be substituted with a residue selected from I, V, L, F, C, M, A, G, T, and W (all of which are more hydrophobic than S). Combinations of mutations (preferably, substitutions) that individually increase hydrophobicity and individually decrease hydrophobicity are also within the scope of the present disclosure, provided that the sum hydrophobicity of all residues in the sequence / region is increased relative to the corresponding wild-type sequence / region. For the purposes of the present disclosure, hydrophobicity can be measured using the Kyte and Doolittle scale

[13] , see Table 2, "Hydrophilicity Index", as set forth below (larger values indicate greater hydrophobicity).

[0078] Isoleucine (I) 4.5

[0079] Valine (V) 4.2

[0080] Leucine (L) 3.8

[0081] Phenylalanine (F) 2.8

[0082] Cysteine (C) 2.5

[0083] Methionine (M) 1.9

[0084] Alanine (A) 1.8

[0085] Glycine (G) -0.4

[0086] Threonine (T) -0.7

[0087] Tryptophan (W) -0.9

[0088] Serine (S) -0.8

[0089] Tyrosine (Y) -1.3

[0090] Proline (P) -1.6

[0091] Histidine (H) -3.2

[0092] Glutamic acid (E) -3.5

[0093] Glutamine (Q) -3.5

[0094] Aspartic acid (D) -3.5

[0095] Asparagine (N) -3.5

[0096] Lysine (K) -3.9

[0097] Arginine (R)-4.5

[0098] References to the sequence / region of the RSV-F protein of the present disclosure that "corresponds to positions x-y of SEQ ID NO:1" encompass the sequence / region aligned with positions x-y of SEQ ID NO:1 (for the avoidance of doubt, which includes positions x and y). However, in a preferred embodiment, the at least one mutation (as defined throughout the present disclosure) is introduced within positions x-y of SEQ ID NO:1 (likewise, including positions x and y). The alignment can be performed visually or by any well-known algorithm; for example, using the NCBI BLAST algorithm, such as "blastp", for example, using the default settings (available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE=Proteins), or for example, using the "ClustalOmega" algorithm (see, for example,

[14] ), for example, using the default settings; the Clustal Omega algorithm is preferred. The corresponding residue positions (such as positions 55, 215, and 348 of SEQ ID NO:1, etc.) are readily identifiable to the person skilled in the art and can be identified by aligning the amino acid sequences using any well-known method (visually or algorithmically, for example, as detailed above).

[0099] As used herein, the "heptad repeat A" domain ("HRA") refers to positions 149-206 of SEQ ID NO:1, the "heptad repeat B" domain ("HRB") refers to positions 474-523 of SEQ ID NO:1, and the "heptad repeat C" domain ("HRC") refers to positions 53-100 of SEQ ID NO:1.

[0100] The RSV-F protein of the present disclosure is preferably an antigen (or, in other words, is antigenic). Thus, the RSV-F protein of the present disclosure preferably elicits an immune response when administered in vivo, namely against RSV. The immune response can include an antibody response (usually including IgG) and / or a cell-mediated immune response, particularly an antibody response. The immune response will generally recognize the three-dimensional structure of the corresponding wild-type pre-fusion RSV-F, particularly one or more epitopes present on the surface of the protein (exposed to the solvent) in the pre-fusion conformation.

[0101] In view of the fact that the RSV-F protein of the present disclosure can be bound by antibodies AM14, D25, RSB1, and motavizumab (especially AM14, D25, and RSB1, especially AM14), it can also be considered an antigen (or, in other words, antigenic). For example, as measured by SPR, its dissociation constant (K D ) is less than 10 nM, such as 1 pM - 10 nM, as detailed below.

[0102] In the RSV-F protein of the present disclosure, it is contemplated to incorporate natural and non-naturally occurring amino acids, although natural amino acids are preferred.

[0103] Generally, the RSV-F protein of the present disclosure elicits a pre-fusion RSV-F specific antibody response against RSV in vivo, such as an IgG antibody response (see, for example, Example 10, Example 11, and Example 13).

[0104] Generally, the RSV-F protein of the present disclosure elicits a neutralizing antibody response against RSV in vivo, such as against RSV A (see, for example, Example 10, Example 11, and Example 13). The neutralizing antibody response can inhibit the replication of RSV in the respiratory system of a subject (such as in the lungs). The neutralizing antibody response can generate protective immunity against RSV in a subject.

[0105] Pre-fusion conformation

[0106] Generally, the RSV-F protein of the present disclosure can be considered to be stabilized in the pre-fusion conformation.

[0107] The pre-fusion conformation of the RSV-F protein of the present disclosure can be confirmed by the binding of a pre-fusion RSV-F specific monoclonal antibody (“pre-fusion mAb”). For example, the RSV-F protein of the present disclosure can be specifically bound by a pre-fusion mAb comprising a light chain and a heavy chain (LC and HC) selected from the group consisting of SEQ ID NO:2 and 3, SEQ ID NO:4 and 5, and SEQ ID NO:8 and 9, respectively. The foregoing are the LC and HC sequences of pre-fusion mAbs AM14, D25, and RSB1, respectively; see, for example, [15,16,17].

[0108] (One or more) specific binding (or lack thereof) of the pre-fusion mAb can be determined by surface plasmon resonance (“SPR”) or biolayer interferometry (“BLI”), however SPR is preferred. SPR can be performed using a BIACORE system, preferably as in the Examples (see subsection “Binding Kinetics Using BIACORE”). Generally, the RSV-F protein of the present disclosure can be specifically bound by any of the above pre-fusion mAbs, as measured by SPR, with a dissociation constant (K D ) less than 10 nM, such as 1 pM - 10 nM; particularly less than 1 nM (1000 pM), such as 1 - 1000 pM.

[0109] For determining the pre-fusion conformation by antibody binding, AM14 is preferred. Unlike other pre-fusion mAbs, AM14 is specific for RSV-F in the pre-fusion conformation when in the intact trimer. The antibody motavizumab (see, e.g.,

[18] ) was also used in the Examples (LC and HC of SEQ ID NOs: 6 and 7, respectively), but also binds the post-fusion conformation and is thus not preferred for confirming the pre-fusion conformation.

[0110] In a specific embodiment, the RSV-F protein of the present disclosure can be specifically bound by a pre-fusion mAb comprising the LC and HC according to SEQ ID NOs: 2 and 3, respectively (or alternatively defined as antibody AM14), as measured by SPR, with a K D less than 1000, 900, 800, 700, 650 or 600 pM; or, in certain embodiments, less than 550 pM; or, in certain embodiments, less than 100, 90, 80, 70, 60, 50 or 40 pM; or, in certain embodiments, less than 35 pM. Lower K D (such as those K D ) are preferred embodiments. By way of example, RSV-F proteins according to the present disclosure named F216, F217, F224 and F225 are specifically bound by this mAb, as measured by SPR, with K D of 598, 546, 37.8 and 30.2 pM, respectively (see, e.g., Example 4, Figure 10 ). The RSV-F protein of the present disclosure can be specifically bound by a pre-fusion mAb comprising the LC and HC according to SEQ ID NOs: 2 and 3, respectively (or alternatively defined as antibody AM14), as measured by SPR, with a K DRanging from 1 - 1000, 1 - 900, 1 - 800, 1 - 700, 1 - 650, 1 - 600, 1 - 550, 1 - 100, 1 - 90, 1 - 80, 1 - 70, 1 - 60, 1 - 50, or 1 - 40 pM; such as 10 - 1000, 10 - 900, 10 - 800, 10 - 700, 10 - 650, 10 - 600, 10 - 550, 10 - 100, 10 - 90, 10 - 80, 10 - 70, 10 - 60, 10 - 50, or 10 - 40 pM; such as 20 - 1000, 20 - 900, 20 - 800, 10 - 700, 20 - 650, 20 - 600, 20 - 550, 20 - 100, 20 - 90, 20 - 80, 20 - 70, 20 - 60, 20 - 50, or 20 - 40 pM. In the foregoing embodiments in this paragraph, the RSV-F protein of the present disclosure is generally assembled in a trimeric form as a homotrimer.

[0111] In a specific embodiment, the RSV-F protein of the present disclosure can be specifically bound by a prefusion mAb comprising an LC and an HC according to SEQ ID NO: 4 and 5, respectively (or alternatively defined as antibody D25), as measured by SPR, with a K D less than 200, 180, 160, 140, or 130 pM; or, in certain embodiments, less than 100, 95, 90, or 85 pM; or, in certain embodiments, less than 80 pM; or, in certain embodiments, less than 70 pM. By way of example, RSV-F proteins according to the present disclosure named F216, F217, F224, and F225 are specifically bound by such an mAb, as measured by SPR, with a K D of 119, 75.2, 67.8, and 83.6 pM, respectively (see, for example, Example 4; Figure 10 ). The RSV-F protein of the present disclosure can be specifically bound by a prefusion mAb comprising an LC and an HC according to SEQ ID NO: 4 and 5, respectively (or alternatively defined as antibody D25), as measured by SPR, with a K Din the range of 1 - 200, 1 - 180, 1 - 160, 1 - 140, 1 - 130, 1 - 100, 1 - 95, 1 - 90, 1 - 85, 1 - 80 or 1 - 70 pM; such as 20 - 200, 20 - 180, 20 - 160, 20 - 140, 20 - 130, 20 - 100, 20 - 95, 20 - 90, 20 - 85, 20 - 80 or 20 - 70 pM; such as 40 - 200, 40 - 180, 40 - 160, 40 - 140, 40 - 130, 40 - 100, 40 - 95, 40 - 90, 40 - 85, 40 - 80 or 40 - 70 pM; such as 50 - 200, 50 - 180, 50 - 160, 50 - 140, 50 - 130, 50 - 100, 50 - 95, 50 - 90, 50 - 85, 50 - 80 or 50 - 70 pM.

[0112] In a specific embodiment, the RSV-F protein of the present disclosure can be specifically bound by a pre-fusion mAb comprising LC and HC according to SEQ ID NOs: 8 and 9 respectively (or differently defined as antibody RSB1), as measured by SPR, with a K D less than 150, 120, 110, 100, 105, 95 or 90 pM; or, in certain embodiments, less than 80, 75 or 70 pM; or, in certain embodiments, less than 60, 55 or 50 pM; or, in certain embodiments, less than 45 pM. For example, the RSV-F proteins according to the present disclosure named F216, F217, F224 and F225 are specifically bound by such mAb, as measured by SPR, with a K D of 85.6, 67.6, 40.4 and 46.5 pM respectively (see, for example, Example 4, Figure 10 ). The RSV-F protein of the present disclosure can be specifically bound by a pre-fusion mAb comprising LC and HC according to SEQ ID NOs: 6 and 7 respectively (or differently defined as antibody RSB1), as measured by SPR, with a K D in the range of 1 - 150, 1 - 120, 1 - 110, 1 - 100, 1 - 105, 1 - 95, 1 - 80, 1 - 75, 1 - 70, 1 - 60, 1 - 55, 1 - 50 or 1 - 45 pM; such as 10 - 150, 10 - 120, 10 - 110, 10 - 100, 10 - 105, 10 - 95, 10 - 80, 10 - 75, 10 - 70, 10 - 60, 10 - 55, 10 - 50 or 10 - 45 pM; such as 20 - 150, 20 - 120, 20 - 110, 20 - 100, 20 - 105, 20 - 95, 20 - 80, 20 - 75, 20 - 70, 20 - 60, 20 - 55, 20 - 50 or 20 - 45 pM.

[0113] In a preferred embodiment, the RSV-F protein of the present disclosure is specifically bound by:

[0114] (i) a pre-fusion mAb comprising an LC and an HC according to SEQ ID NO: 2 and 3, respectively (or alternatively defined as antibody AM14), having a K D less than 1000, 900, 800, 700 or less than 650 pM (such as 1 - 1000, 1 - 900, 1 - 800, 1 - 700 or 1 - 650 pM);

[0115] (ii) a pre-fusion mAb comprising an LC and an HC according to SEQ ID NO: 4 and 5, respectively (or alternatively defined as antibody D25), having a K D less than 300, 250, 200, 150 or less than 130 pM; optionally less than 100 or 80 pM (such as 1 - 300, 1 - 250, 1 - 200, 1 - 150 or 1 - 130 pM; optionally 1 - 100 or 1 - 80 pM); and / or

[0116] (iii) a pre-fusion mAb comprising an LC and an HC according to SEQ ID NO: 8 and 9, respectively (or alternatively defined as antibody RSB1), having a K D less than 200, 150, 100 or 90 pM (such as 1 - 200, 1 - 150, 1 - 100 or 1 - 90 pM);

[0117] wherein the K according to (i)-(iii) D is measured by SPR. Preferably, the RSV-F protein of the present disclosure meets 2 of the criteria (i), (ii) and (iii), or more preferably all 3. For example, proteins F216 and F217 meet all of the said criteria (see, for example, Example 4, Figure 10 ), where F217 meets the optional criteria specified in (ii). Optionally, such RSV-F proteins can be bound by an antibody comprising an LC and an HC according to SEQ ID NO: 6 and 7, respectively (or alternatively defined as motavizumab), as measured by SPR, having a K D less than 200, 150, 100 or less than 80 pM (such as 1 - 200, 1 - 150, 1 - 100 or 1 - 80 pM).

[0118] In a preferred embodiment, the RSV-F protein of the present disclosure is specifically bound by:

[0119] (iv) a pre-fusion mAb comprising an LC and an HC according to SEQ ID NO: 2 and 3, respectively (or alternatively defined as antibody AM14), having a K DLess than 200, 150, 100, 80, 60 or 40 pM (such as 1 - 200, 1 - 150, 1 - 100, 1 - 80, 1 - 60 or 1 - 40 pM);

[0120] (v) A pre - fusion mAb comprising an LC and an HC according to SEQ ID NO:4 and 5 respectively (or alternatively defined as antibody D25), the K D Less than 200, 150, 100, 90 or 85 pM; optionally less than 70 pM (such as 1 - 200, 1 - 150, 1 - 100, 1 - 90 or 1 - 85 pM; optionally 1 - 70 pM); and / or

[0121] (vi) A pre - fusion mAb comprising an LC and an HC according to SEQ ID NO:8 and 9 respectively (or alternatively defined as antibody RSB1), the K D Less than 200, 100, 80, 60 or 50 pM (such as 1 - 200, 1 - 100, 1 - 80, 1 - 60 or 1 - 50 pM);

[0122] wherein the K according to (iv) - (vi) D is measured by SPR. Preferably, the RSV - F protein of the present disclosure meets 2 of the criteria (iv), (v) and (vi), or more preferably all 3. For example, proteins F224 and F225 meet all the said criteria (see, for example, Example 4, Figure 10 ), where F224 meets the optional criterion set forth in (v). Optionally, such an RSV - F protein can be bound by an antibody comprising an LC and an HC according to SEQ ID NO:6 and 7 respectively (or alternatively defined as motavizumab), as measured by SPR, the K D Less than 40 pM (such as 1 - 40 pM).

[0123] Generally, the RSV-F protein of the present disclosure can be bound by a prefusion mAb (in particular, any of those defined above) for a period of, for example, at least: 24 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks, 7 weeks, or 8 weeks; for example, where the RSV-F protein is stored in buffer at 4 °C or 25 °C for the said (one or more) periods and then assayed to determine the presence of specific binding by a prefusion mAb (in particular AM14 or D25) or an antigen-binding fragment thereof (such as its Fab fragment). The binding over a period of time can be determined, for example, by SPR or BLI. The buffer can be a HEPES buffer, for example 20 mM HEPES containing 150 mM NaCl. Thermal stability can also be evaluated (for example using Nano-DSF, for example as carried out in the Examples). Aggregation of the protein can also be evaluated (for example by high performance liquid chromatography (“HPLC”), for example as carried out in the Examples).

[0124] In addition to the above, the RSV-F protein of the present disclosure can also be bound by an antibody comprising an LC and an HC according to SEQ ID NOs: 6 and 7 respectively (or otherwise defined as motavizumab), as measured by SPR, the K D less than 200, 180, 160, 140, or 120 pM; or, in certain embodiments, less than 110, 100, or 95 pM; or, in certain embodiments, less than 80, 70, 60, or 55 pM; or, in certain embodiments, less than 50, 45, or 40 pM. By way of example, RSV-F proteins according to the present disclosure named F216, F217, F224, and F225 are specifically bound by such mAb, as measured by SPR, the K D being 74.8, 117, 38.6, and 52.8 pM respectively (see, for example, Example 4, Figure 10 ). The RSV-F protein of the present disclosure can be specifically bound by a prefusion mAb (or otherwise defined as motavizumab) comprising an LC and an HC according to SEQ ID NOs: 6 and 7 respectively, as measured by SPR, the K Din the range of 1 - 200, 1 - 180, 1 - 160, 1 - 140, 1 - 120, 1 - 110, 1 - 100, 1 - 95, 1 - 80, 1 - 70, 1 - 55, 1 - 50, 1 - 45 or 1 - 40 pM; such as 10 - 200, 10 - 180, 10 - 160, 10 - 140, 10 - 120, 10 - 110, 10 - 100, 10 - 95, 10 - 80, 10 - 70, 10 - 55, 10 - 50, 10 - 45 or 10 - 40 pM, such as 20 - 200, 20 - 180, 20 - 160, 20 - 140, 20 - 120, 20 - 110, 20 - 100, 20 - 95, 20 - 80, 20 - 70, 20 - 55, 20 - 50, 20 - 45 or 20 - 40 pM.

[0125] In an alternative and more preferred method of mAb binding, the pre - fusion conformation of the RSV - F protein of the present disclosure can be confirmed by single - particle analysis of cryo - electron microscopy ("cyro - EM" - see, for example, Example 4, Figure 13 - Figure 16 ) preferably when the protein is complexed with the antigen - binding fragment of the pre - fusion mAb. Preferably, such cryo - EM includes the following steps:

[0126] Complex the RSV - F protein of the present disclosure with the antigen - binding fragment of the pre - fusion mAb (such as a Fab fragment) (preferably the antigen - binding fragment of AM14, preferably the Fab fragment of AM14) to form a complex;

[0127] Isolate (e.g., by gel filtration) and concentrate the complex;

[0128] Deposit the complex on an electron microscopy grid and vitrify the complex and the grid (e.g., by plunging into liquid ethane for freezing);

[0129] Image by electron microscopy; and

[0130] Resolve the structure of the complex by single - particle analysis.

[0131] More preferably, such cryo - EM is performed as in the examples (see subsection "Cryo - electron microscopy of RSV - F designs F21, F216, and F224").

[0132] Residues containing hydrogen bond donor and / or acceptor moieties in the side chain

[0133] The RSV-F protein of the present disclosure comprises (according to the first independent aspect) or may comprise (according to the third or fourth independent aspect) at least one mutation relative to the wild type in the region corresponding to positions 217-239 of SEQ ID NO:1; wherein the at least one mutation is introduced by substitution or insertion into a residue that contains an H-bond donor and / or acceptor moiety in the side chain.

[0134] Preferably, the region in which the at least one mutation is located comprises or consists of an α-helix. Preferably, the residue forms a hydrogen bond with other residues in the RSV-F protein; preferably, wherein the other residues are within the region corresponding to positions 239-254 of SEQ ID NO:1; more preferably, wherein the other residue is at position 250 of SEQ ID NO:1; more preferably, wherein the other residue is Y250 (wild type) or D250, even more preferably Y250.

[0135] Both natural and non-natural residues that contain an H-bond donor and / or acceptor moiety in the side chain are within the scope of the present disclosure. Natural residues that contain such moieties in the side chain include R, K, W (containing an H-bond donor moiety), D, E (containing an H-bond acceptor moiety), N, Q, H, S, T, and Y (containing both an H-bond donor and acceptor moiety). Thus, as used herein, the term "residue that contains an H-bond donor and / or acceptor moiety in the side chain" encompasses residues that contain in the side chain: (i) an H-bond donor moiety, (ii) an H-bond acceptor moiety, (iii) both an H-bond donor moiety and an H-bond acceptor moiety (i.e., separate donor and acceptor moieties), and (iv) an H-bond donor and acceptor moiety (i.e., a moiety capable of acting as both a donor and an acceptor). "Residue that contains an H-bond donor and / or acceptor moiety..." may be used interchangeably with "residue that contains an H-bond donor and / or acceptor atom...".

[0136] As noted above, "corresponding to..." encompasses the sequence / region of the RSV-F protein of the present disclosure aligned with the corresponding wild-type sequence / region. Thus, the RSV-F protein of the present disclosure may comprise: at least one mutation relative to SEQ ID NO:1 in the region of the protein aligned with positions 217-239 of SEQ ID NO:1 (preferably which forms an α-helix), wherein the at least one mutation is introduced by substitution or insertion into a residue that contains an H-bond donor and / or acceptor moiety in the side chain. However, in a preferred embodiment, the RSV-F protein of the present disclosure comprises: at least one mutation relative to SEQ ID NO:1 within positions 217-239 of SEQ ID NO:1, wherein the at least one mutation is introduced by substitution or insertion into a residue that contains an H-bond donor and / or acceptor moiety in the side chain.

[0137] In the wild-type RSV-F protein, an α5 helix is formed at positions 217-239. Without being bound by this theory, introducing an H-bond donor and / or acceptor moiety may result in the formation of H-bonds between residues in the loop corresponding to positions 239-254 of SEQ ID NO:1 within the protomer (i.e., between residues within a single protomer). This H-bond can provide stabilizing interactions that favor the pre-fusion conformation. This H-bond and / or the resulting stabilizing interactions can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation. This stabilization mechanism is in contrast to reference

[19] , which introduced F or L residues into the 217-239 region (among other substitutions). F and L residues can readily participate in van der Waals (VDW) contacts, but lack H-bond acceptor and / or donor moieties (such as N or O) in their side chains.

[0138] The RSV-F protein of the present disclosure can comprise at least one mutation relative to SEQ ID NO:1 in the region corresponding to positions 217-239 of SEQ ID NO:1 (preferably forming an α helix), wherein the at least one mutation introduces a residue selected from K, R, N, W, D, E, Q, H, S, T, and Y into the region by substitution or insertion. Residues containing an H-bond donor moiety in their side chains (i.e., K, R, W, N, Q, H, S, T, and Y) are generally preferred; among such residues, K, R, Q, and N are preferred (more preferably K or R, even more preferably K). Substitution at position 228 of SEQ ID NO:1 is generally preferred.

[0139] The at least one mutation can comprise, consist of, or be composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 substitutions or insertions (preferably substitutions) relative to positions 217-239 of SEQ ID NO:1; in particular, only 1, 2, 3, 4, 5, 6, 7, or 8 such substitutions or insertions (preferably substitutions), in particular only 1, 2, 3, 4, or 5 such substitutions or insertions (preferably substitutions), in particular only 1 or 2 such substitutions or insertions (preferably substitutions), preferably only 1 such substitution or insertion (preferably substitution).

[0140] In the RSV-F protein of the present disclosure, the region corresponding to positions 217-239 of SEQ ID NO:1 (preferably forming an α helix) can have at least 50%, 60%, 70%, 80% sequence identity with positions 217-239 of SEQ ID NO:1, or preferably at least 85%, 90%, or 95% sequence identity.

[0141] In some embodiments of the RSV-F protein of the present disclosure, one or more wild-type residues (especially 1 or 2 residues, especially only 1 residue) in the region corresponding to positions 220-235 (especially positions 227-232, especially positions 228-232) of SEQ ID NO:1 can be replaced with residues selected from: K, R, N, W, D, E, Q, H, S, T, and Y (e.g., K, R, N, W, Q, H, S, T, and Y; preferably K, R, Q, and N; more preferably K and R; even more preferably K).

[0142] In a preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 228 (N) of SEQ ID NO:1 with K, R, Q, or N; or a substitution with K, R, or Q (preferably K or R, preferably K), and / or a substitution at position 232 (E) of SEQ ID NO:1 with N. The N at position 232 also provides an H-bond donor moiety that is within a suitable distance to form a potential H-bond with Y250 (see Figure 25 ). In a preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 228 (N) of SEQ ID NO:1 with K, R, Q, or N; or a substitution with K, R, or Q. In a more preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 228 (N) of SEQ ID NO:1 with K or R. In an even more preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 228 (N) of SEQ ID NO:1 with K. Such preferred substitutions at positions 228 and / or 232 can be the only mutations that introduce residues containing H-bond donor and / or acceptor moieties within the region corresponding to positions 217-239 of SEQ ID NO:1; and optionally the only mutations within the region corresponding to positions 217-239 of SEQ ID NO:1. In the foregoing embodiments in this paragraph (where position 228 is substituted), preferably, position 232 has the wild-type residue (E) of SEQ ID NO:1, or is substituted with D (also a negatively charged residue). The E or D at position 232 can contribute to providing the tertiary cation-pi-anion interaction discussed in the following paragraph. In the foregoing embodiments in this paragraph, preferably, position 250 has the wild-type residue (Y) of SEQ ID NO:1, or is substituted with D.

[0143] As detailed in Example 6, the minimal substitution screen conducted by the inventors revealed that the N228K substitution was able to achieve pre-fusion RSV-F alone (see Figure 19 ; designed F310). Without being bound by this theory, based on three-dimensional structure analysis, replacing N with K at position 228 appears to result in the formation of an H-bond with Y250 on the same protomer (see Figure 25, the dashed line indicates a hydrogen bond). The H-bond can stabilize Y250 to form a tertiary cation-pi-anion interaction between E232, Y250, and R235 (E232 and Y250 are on one protomer, and R235 is on an adjacent protomer). E, Y, and R are one of the major triads of this tertiary cation-pi-anion interaction (see, for example,

[20] ). In addition, residues at position 228 with other H-bond donors in the side chain (such as R) can also provide this stabilizing H-bond with Y250. Q also provides an H-bond donor moiety with a side chain large enough to form a potential H-bond with Y250. In addition, based on the proximity and orientation of the E232 side chain (see Figure 25 ), substitution with, in particular, N can also provide a stabilizing hydrogen bond with Y250.

[0144] In all of the foregoing embodiments in this subsection, optionally, the RSV-F protein of the present disclosure may comprise a substitution of Y at position 250 of SEQ ID NO:1 with D. The Y250D substitution can strengthen the trans-protomer interaction with R235 (wild-type residue) by forming a salt bridge between the two residues. For an example of an engineered trans-protomer salt bridge between D and R residues to stabilize the multimeric protein in the pre-fusion conformation, see

[21] (Figure 1; D961–R765 salt bridge). In addition, D contains an H-bond acceptor moiety, so the Y250D substitution will maintain the preferred hydrogen bond between positions 250 and 228 (in a preferred embodiment, comprising an N228K or N228R substitution, more preferably an N228K substitution).

[0145] Generally, the mutations detailed throughout this subsection (preferably substitutions, preferably such substitutions at positions 228 and / or 232, preferably such substitutions at position 228) can provide core stabilization in the F1 domain (positions 137-513 of SEQ ID NO:1), close to the heptad repeat A ("HRA") domain and the antibody binding site ("site ”). Generally, the mutations detailed throughout this subsection (preferably substitutions, preferably such substitutions at positions 228 and / or 232, preferably such substitutions at position 228) can provide H-bonds with Y250, for example, stabilizing Y250 to provide a tertiary cation-pi-anion interaction between the following positions in different RSV-F protomers: (i) position 232 (preferably E232 as in the wild type, or D232 if substituted), (ii) Y250, and (ii) R235. Such core stabilization, H-bonds, and / or tertiary anion-pi-cation interactions can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation. Generally, the mutations detailed throughout this subsection (preferably substitutions, preferably substitutions at positions 228 and / or 232, preferably substitutions at position 228) can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation.

[0146] Further mutations: (a)

[0147] The RSV-F protein of the present disclosure may further comprise (according to all independent aspects of the present disclosure):

[0148] (ai) at least one mutation relative to the wild type in the region corresponding to positions 38-60 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 38-60 of SEQ ID NO:1; and / or

[0149] (aii) at least one mutation relative to the wild type in the region corresponding to positions 296-318 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 296-318 of SEQ ID NO:1, and / or residues selected from M, F, I, and V are introduced into the region by substitution or insertion.

[0150] In the embodiment of (a) below, the mutation according to (ai) is preferred.

[0151] Preferably, the region where the at least one mutation according to (ai) is located comprises or consists of a beta-sheet, and the at least one mutation increases the hydrophobicity of the beta-sheet relative to the wild-type beta-sheet (i.e., positions 38-60 of SEQ ID NO:1). Preferably, the region where the at least one mutation according to (aii) is located comprises or consists of a beta-sheet, and the at least one mutation increases the hydrophobicity of the beta-sheet relative to the wild-type beta-sheet (i.e., positions 296-318 of SEQ ID NO:1).

[0152] As noted above, "corresponding to" encompasses the sequences / regions of the RSV-F proteins of the present disclosure aligned with the corresponding wild-type sequences / regions. Thus, the RSV-F proteins of the present disclosure can comprise: (ai) at least one mutation relative to SEQ ID NO:1 in the region of the protein aligned with positions 38 - 60 of SEQ ID NO:1 (preferably which forms a beta-sheet), wherein the at least one mutation increases the hydrophobicity of the region relative to positions 38 - 60 of SEQ ID NO:1; and / or (aii) at least one mutation relative to SEQ ID NO:1 in the region of the protein aligned with positions 296 - 318 of SEQ ID NO:1 (preferably which forms a beta-sheet), wherein the at least one mutation increases the hydrophobicity of the region relative to positions 296 - 318 of SEQ ID NO:1 and / or introduces a residue selected from M, F, I, and V into the region. However, in a preferred embodiment, the RSV-F proteins of the present disclosure comprise: (ai) at least one mutation relative to SEQ ID NO:1 within positions 38 - 60 of SEQ ID NO:1, wherein the at least one mutation results in an increase in hydrophobicity relative to said positions; and / or (aii) at least one mutation relative to SEQ ID NO:1 within positions 296 - 318 of SEQ ID NO:1, wherein the at least one mutation results in an increase in hydrophobicity relative to said positions and / or introduces a residue selected from M, F, I, and V into said positions.

[0153] In the wild-type RSV-F sequence, positions 38 - 60 and 296 - 318 form two beta-sheets that form at least a portion of a largely hydrophobic pocket at the interface between the F1 domain (positions 137 - 513 of SEQ ID NO:1) and the heptad repeat A ("HRA") domain, see Figure 22 . Without wishing to be bound by theory, increasing the hydrophobicity of one or both of the corresponding beta-sheets in the RSV-F proteins of the present disclosure (relative to wild-type) can provide new energetically favorable van der Waals (VDW) contacts within the largely hydrophobic pocket. Introducing M, F, I, and V into the beta-sheet corresponding to positions 296 - 318 (which have relatively large and / or hydrophobic side chains) can also provide such VDW contacts. Such VDW contacts can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation.

[0154] The at least one mutation according to (ai) may comprise, consist of, or consist essentially of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 mutations (preferably substitutions) relative to positions 38 - 60 of SEQ ID NO:1; particularly only 1, 2, 3, 4, 5, 6, 7, or 8 such mutations (preferably substitutions); particularly only 1, 2, 3, 4, or 5 such mutations (preferably substitutions); particularly only 1 or 2 such mutations (preferably substitutions); preferably only 1 such mutation (preferably substitution).

[0155] The at least one mutation according to (aii) may comprise, consist of, or consist essentially of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 mutations (preferably substitutions) relative to positions 296 - 318 of SEQ ID NO:1; particularly only 1, 2, 3, 4, 5, 6, 7, or 8 such mutations (preferably substitutions); particularly only 1, 2, 3, 4, or 5 such mutations (preferably substitutions); particularly only 1 or 2 such mutations (preferably substitutions); preferably only 1 such mutation (preferably substitution).

[0156] In the RSV-F protein of the present disclosure, the region corresponding to positions 38 - 60 of SEQ ID NO:1 (preferably forming a β-sheet) may have at least 50%, 60%, 70%, 80% sequence identity with positions 38 - 60 of SEQ ID NO:1, or preferably at least 85%, 90%, or 95% sequence identity. In the RSV-F protein of the present disclosure, the region corresponding to positions 296 - 318 of SEQ ID NO:1 (preferably forming a β-sheet) may have at least 50%, 60%, 70%, 80% sequence identity with positions 296 - 318 of SEQ ID NO:1, or preferably at least 85%, 90%, or 95% sequence identity.

[0157] In some embodiments of the RSV-F protein of the present disclosure, one or more S residues in the wild-type beta-sheet at positions 38-60 of SEQ ID NO:1 (e.g., at positions 38, 41, 46, and / or 55) can be replaced with a residue that is more hydrophobic than S (e.g., I, V, L, F, C, M, A, G, T, or W). For example, positions 38, 41, 46, and / or 55 of SEQ ID NO:1 can be replaced with T, C, V, I, or F, particularly T, C, or V, preferably T. Additionally or alternatively, in some embodiments, the RSV-F protein of the present disclosure can comprise a substitution at position 301 with a residue selected from M, F, and I; and / or a substitution at position 303 with a residue selected from V, M, F, and I; in particular, such substitutions are present at both positions 301 and 303. In the wild-type, the V301 and L303 side chains point into the largely hydrophobic pocket discussed above (see Figure 22 ). Thus, without being bound by theory, introducing relatively large and / or hydrophobic side chains at these positions can, in particular, provide energetically favorable VDW contacts within the pocket.

[0158] In a specific embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 55 (S) of SEQ ID NO:1 with a more hydrophobic residue (e.g., I, V, L, F, C, M, A, G, T, or W, which is more hydrophobic than the S at wild-type position 55). Optionally, in such embodiments, the RSV-F protein of the present disclosure can comprise a substitution at position 301 with a residue selected from M, F, and I; and / or a substitution at position 303 with a residue selected from V, M, F, and I; in particular, such substitutions are present at both positions 301 and 303.

[0159] In a preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution of T, C, V, I, or F for S at position 55 of SEQ ID NO:1. In a more preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution of T, C, or V for S at position 55 of SEQ ID NO:1. In a more preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution of T or V for S at position 55 of SEQ ID NO:1. In an even more preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution of T for S at position 55 of SEQ ID NO:1. Such preferred substitutions at position 55 may be the only mutations according to (ai); and optionally the only mutations in the region corresponding to positions 38-60 of SEQ ID NO:1. Such preferred substitutions at position 55 may be the only mutations according to (ai), where (aii) no mutations are present; and optionally the only mutations in the region corresponding to positions 38-60 of SEQ ID NO:1.

[0160] As detailed in Example 6, the minimal substitution screen conducted by the inventors revealed that the S55T mutation may be a driver of the pre-fusion conformation (designated F308). Without wishing to be bound by this theory, substituting T for S at position 55 provides a slightly larger residue which (from computer-simulated three-dimensional structure analysis, see Figure 22 ) appears to fit well into the hydrophobic pocket discussed above without creating significant steric clashes. Additionally, the addition of the CH3 group of T appears to provide new energetically favorable VDW contacts of the type discussed above. Additionally, alternative substitutions provided by the ROSETTA software for position 55 include C and V (based on allowing all amino acids (no evolutionary constraints), using an energy threshold of 0.0, -0.1, or -0.5).

[0161] Generally, a mutation according to (a) (preferably a substitution, preferably such a substitution at position 55 as detailed above) can stabilize the interface between the F1 domain (positions 137 - 513 of SEQ ID NO:1) and the heptad repeat A ("HRA") domain. This stabilization can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation. Generally, a mutation according to (a) (preferably such a substitution, preferably the substitution at position 55 as detailed above) can provide an energetically favorable VDW contact within the hydrophobic pocket of RSV-F, at the interface between the F1 domain and the HRA domain. Such contact can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation. Generally, a mutation according to (a) (preferably a substitution, preferably the substitution at position 55 as detailed above) can inhibit the refolding of the HRA and HRC domains. This refolding can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation. Generally, a mutation according to (a) (preferably a substitution, preferably the substitution at position 55 as detailed above) can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation.

[0162] Further mutations: (b)

[0163] In addition to or in contrast to the above (a), the RSV-F protein of the present disclosure can further comprise (in accordance with all independent aspects of the present disclosure):

[0164] At least one mutation relative to the wild type in the region corresponding to positions 208 - 216 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 208 - 216 of SEQ ID NO:1, and / or introduces a P residue into the region by substitution or insertion.

[0165] Preferably, the region where the at least one mutation according to (b) is located comprises or consists of a loop (more preferably a loop connecting two α-helices), and the at least one mutation increases the hydrophobicity of the loop relative to the wild type hinge (i.e., positions 208 - 216 of SEQ ID NO:1), and / or introduces at least one P residue into the hinge. As mentioned herein, a "loop" can also be referred to as a "loop region" or a "flexible loop", or, if a part of the protein rotates around the loop during a conformational change (especially as in the case of positions 208 - 216), it is called a "hinge loop", "hinge", or "hinge region".

[0166] As mentioned above, "corresponding to" encompasses the sequences / regions of the RSV-F proteins of the present disclosure aligned with the corresponding wild-type sequences / regions. Thus, in some embodiments, the RSV-F proteins of the present disclosure may comprise: (b) at least one mutation relative to SEQ ID NO:1 in the protein sequence aligned with positions 208-216 of SEQ ID NO:1 (preferably forming a loop, more preferably a loop connecting two α-helices), wherein the at least one mutation increases the hydrophobicity of the region relative to positions 208-216 of SEQ ID NO:1, and / or introduces a P residue into the region. However, in a preferred embodiment, the RSV-F proteins of the present disclosure comprise: (b) at least one mutation relative to SEQ ID NO:1 within positions 208-216 of SEQ ID NO:1, wherein the at least one mutation results in an increase in hydrophobicity relative to said positions, and / or introduces a P residue into said positions.

[0167] In the wild-type RSV-F sequence, positions 208-216 form a loop connecting two α-helices (α4 helix and α5 helix in the wild-type), see Figure 23 . Without being bound by this theory, increasing the hydrophobicity of the loop and / or introducing a P residue into the loop may stabilize or rigidify the loop, and / or facilitate packing away from the RSV-F surface. Such stabilization, rigidification, and / or packing may inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation (in particular, by inhibiting the relative movement of two α-helices adjacent to the loop, generally the α4 helix and α5 helix of RSV-F).

[0168] The at least one mutation according to (b) may comprise or consist of 1, 2, 3, 4, 5, 6, 7, or 8 substitutions or insertions (preferably substitutions) relative to positions 208-216 of SEQ ID NO:1; in particular only 1, 2, 3, or 4 such substitutions or insertions (preferably substitutions), in particular only 1, 2, or 3 such substitutions or insertions (preferably substitutions), in particular only 1 or 2 such substitutions or insertions (preferably substitutions), preferably only 1 such substitution or insertion (preferably substitution).

[0169] In the RSV-F proteins of the present disclosure, the region corresponding to positions 38-60 of SEQ ID NO:1 (preferably forming a loop, more preferably a loop connecting two α-helices) may have at least 50% or 60% sequence identity with positions 208-216 of SEQ ID NO:1, or preferably at least 75% or 85% sequence identity.

[0170] In some embodiments of the RSV-F protein of the present disclosure, one or more S residues in the wild-type loop at positions 208-216 of SEQ ID NO:1 (e.g., at positions 211, 213, and / or 215) can be replaced with a residue more hydrophobic than S (e.g., I, V, L, F, C, M, A, G, T, or W). For example, the wild-type residues at positions 211, 213, and / or 215 of SEQ ID NO:1 can be replaced with an A residue or a P residue, preferably A.

[0171] In a preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 215 (S) of SEQ ID NO:1 with A, P, V, I, or F. In a preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 215 (S) of SEQ ID NO:1 with A, V, I, or F. In a more preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 215 (S) of SEQ ID NO:1 with A or P. In an even more preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 215 (S) of SEQ ID NO:1 with A. Such preferred substitutions at position 215 can be the only mutations according to (b); and optionally the only mutations in the region corresponding to positions 208-216 of SEQ ID NO:1.

[0172] As detailed in Example 6, the minimal substitution screen conducted by the inventors revealed that the S215A mutation may be a driver of the pre-fusion conformation (designated F309). Without being bound by this theory, removing the hydrophilic OH group (since S is replaced with A) may favor the stacking and rigidity of this loop (see Figure 23 ). Additionally, the A residue at position 215 can provide energetically favorable VDW contacts with residues at positions 79, 206 (I residue in the wild-type), L203, and / or T219. Such stacking, rigidification, and / or VDW contacts can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation (in particular, inhibit the relative movement of two α-helices adjacent to this loop, generally the α4 and α5 helices of RSV-F, or differently defined as inhibit the refolding of the HRC domain and the HRA domain). Additionally, the side chains of P, V, I, or F can also reduce the conformational freedom of this loop and thus also favor the stacking and rigidification of this loop.

[0173] Generally, a mutation according to (b) (preferably such a substitution, preferably such a substitution at position 215 as detailed above) can stabilize or rigidify the loop corresponding to positions 208 - 216 of SEQ ID NO:1. This stabilization or rigidification can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation (in particular, by inhibiting the relative movement of two α-helices adjacent to the loop (generally the α4 helix and the α5 helix of RSV-F)). Generally, such a mutation according to (b) (preferably such a substitution, preferably such a substitution at position 215 as detailed above) can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation (in particular, by inhibiting the relative movement of two α-helices adjacent to the loop (generally the α4 helix and the α5 helix of RSV-F), or alternatively defined as by inhibiting the refolding of the HRC structure and the HRA domain).

[0174] Further mutations: (c)

[0175] In addition to or in contrast to the above (a) and / or (b), the RSV-F protein of the present disclosure may further comprise (in accordance with all independent aspects of the present disclosure):

[0176] At least one mutation relative to the wild type in the region corresponding to positions 345 - 352 of SEQ ID NO:1, wherein the at least one mutation introduces a glycosylation site into the region by substitution or insertion.

[0177] Preferably, the region where the at least one mutation according to (c) is located comprises a β-sheet and a loop (and optionally, at least a part of other β-sheets), and the at least one mutation introduces a glycosylation site into the region.

[0178] As described above, "corresponding to..." encompasses the sequences / regions of the RSV-F proteins of the present disclosure aligned with the corresponding wild-type sequences / regions. Thus, in some embodiments, the RSV-F proteins of the present disclosure can comprise: (c) at least one mutation relative to SEQ ID NO:1 in the region of the protein aligned with positions 345-352 of SEQ ID NO:1 (preferably comprising a beta-sheet and a loop), wherein the at least one mutation introduces a glycosylation site into the region. However, in a preferred embodiment, the RSV-F proteins of the present disclosure comprise: (c) at least one mutation relative to SEQ ID NO:1 within positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces a glycosylation site into said positions. To avoid doubt, in the wild-type, positions 348-352 of SEQ ID NO:1 form a beta-sheet, positions 346-347 of SEQ ID NO:1 form a loop, and position 345 is the C-terminal residue of another beta-sheet.

[0179] Preferably, the at least one mutation according to (c) results in glycosylation of residues within the region (preferably comprising a beta-sheet and a loop); or alternatively defined as, the at least one mutation according to (c) results in the introduction of a glycan linked to a residue within the region (preferably comprising a beta-sheet and a loop).

[0180] Without wishing to be bound by this theory, glycosylation of positions within the above region can provide a stabilizing trans-protomer interaction with a charged patch at approximately positions 416-422 of SEQ ID NO:1 (especially K419; see Figure 24), thereby inhibiting, at least partially inhibiting, or completely inhibiting the transition of RSV-F from the pre-fusion to the post-fusion conformation. As discussed below, this interaction can be maintained or enhanced by introducing further mutations at positions 416-422 of SEQ ID NO:1.

[0181] The at least one mutation according to (c) can comprise, consist of, or consist essentially of one or more substitutions or insertions (preferably substitutions) relative to positions 345-352 of SEQ ID NO:1; especially only 1, 2, or 3 such substitutions or insertions (preferably substitutions), especially only 1 or 2 such mutations (preferably substitutions), especially only 1 such substitution or insertion (preferably substitution).

[0182] In the RSV-F proteins of the present disclosure, the region corresponding to positions 345-352 of SEQ ID NO:1 (preferably comprising a beta-sheet and a loop) can have at least 50% or 60% sequence identity with positions 345-352 of SEQ ID NO:1, or preferably at least 75% or 85% sequence identity.

[0183] Glycosylation sites / glycosylation can be introduced into this region (preferably comprising a β-sheet and a loop) by introducing at least one N residue (resulting in N-linked glycosylation) or at least one S and / or T residue (resulting in O-linked glycosylation) by mutation, or the glycan can be linked to this region. Preferably, at least one N residue is introduced by substitution, resulting in the generation of an NXT or NXS motif (as required for N-linked glycosylation), where X is any amino acid other than P (as required for N-linked glycosylation).

[0184] Generally, the glycosylation / glycan will comprise a core structure containing or consisting of N-acetylglucosamine (GlcNAc). Generally, the glycosylation / glycan will comprise GlcNAc or consist of it.

[0185] In a preferred embodiment of the RSV-F protein of the present disclosure, the glycosylation site / glycosylation is introduced into the β-sheet corresponding to positions 348-352 of SEQ ID NO:1 by mutation, or the glycan is linked to the residues in the β-sheet. In an even more preferred embodiment of the RSV-F protein of the present disclosure, the glycosylation site / glycosylation is introduced into the said β-sheet by substituting the S at position 348 of SEQ ID NO:1 with N, or the glycan is linked to the said β-sheet. The glycosylation site can be conserved by maintaining the wild-type residue (S) at position 350 or substituting S350 with T. Such a preferred substitution at position 348 can be the only mutation according to (c); and optionally the only mutation in the region corresponding to positions 345-352 of SEQ ID NO:1.

[0186] As detailed in Example 6, the minimal substitution screen conducted by the inventors revealed that the S348N mutation may be a driving factor for the pre-fusion conformation (designated F311). In addition, cryo-EM studies of designs F216 and F225 (see, for example, Example 5) revealed the presence of a glycan linked to an N residue at position 348 (determined by observing additional electron density protruding from the N348 position). This glycan may form a trans-protomer hydrogen bond with a charged local region at approximately positions 416-422 of SEQ ID NO:1 (especially the K419 position) (see Figure 24B ).

[0187] In a specific embodiment, the RSV-F protein of the present disclosure further comprises at least one mutation (preferably a substitution) in the region corresponding to positions 416-422 of SEQ ID NO:1 (preferably forming a loop), wherein the at least one mutation increases the negative charge of the region (e.g., by introducing D and / or E residues). Preferably, the at least one mutation is a substitution of the residue at position 419 of SEQ ID NO:1 with D or E. In an even more preferred embodiment, the at least one mutation is a substitution of the residue at position 419 of SEQ ID NO:1 with D (as in the design of F216, see Figure 24A ). Such mutations in the region corresponding to positions 416-422 of SEQ ID NO:1 (preferably forming a loop) can result in cross-protomer interactions, thereby contributing to the inhibition of the transition of RSV-F from the pre-fusion to the post-fusion conformation.

[0188] Generally, the mutation according to (c) (preferably such a substitution, preferably such a substitution at position 348 as detailed above) can stabilize or rigidify the loop region corresponding to positions 346-347 of SEQ ID NO:1 in the F1 domain. This stabilization or rigidification can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation. Generally, the mutation according to (c) (preferably such a substitution, preferably such a substitution at position 348 as detailed above) can provide cross-protomer interactions (such as hydrogen bonds). Such cross-protomer interactions can occur with one or more charged residues, including, for example, the residue at position 419 of SEQ ID NO:1 (e.g., the K, E, or D residue at position 419). Such cross-protomer interactions can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation. Generally, the mutation according to (c) (preferably such a substitution, preferably such a substitution at position 348 as detailed above) can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation.

[0189] Further mutations: (d)

[0190] In addition to or in contrast to the above (a) and / or (b), the RSV-F protein of the present disclosure may further comprise (in accordance with all independent aspects of the present disclosure):

[0191] At least one mutation relative to the wild type in the region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces at least one residue selected from N, D, F, H, K, L, Q, R, T, W, and Y into the region by substitution or insertion.

[0192] Preferably, the region in which the at least one mutation according to (d) is located comprises a beta-sheet and a loop (and optionally, at least a portion of other beta-sheets). More preferably, the at least one mutation according to (d) is introduced into the beta-sheet corresponding to positions 348-352 of SEQ ID NO:1.

[0193] As noted above, "corresponding to..." encompasses two aligned sequences / regions (one being the sequence / region of the RSV-F protein of the present disclosure and the other being the sequence / region of the wild type). Thus, in some embodiments, the RSV-F protein of the present disclosure may comprise: (d) at least one mutation relative to SEQ ID NO:1 in the region of the protein aligned with positions 345-352 of SEQ ID NO:1 (preferably which comprises a beta-sheet and a loop), wherein the at least one mutation introduces at least one residue selected from D, F, H, K, L, N, Q, R, T, W, and Y into the region. However, in a preferred embodiment, the RSV-F protein of the present disclosure comprises: (d) at least one mutation relative to SEQ ID NO:1 within positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces at least one residue selected from D, F, H, K, L, N, Q, R, T, W, and Y into the said positions.

[0194] The at least one mutation according to (d) may comprise or consist of 1, 2, 3, 4, 5, 6, 7, or 8 substitutions or insertions (preferably substitutions) relative to positions 345-352 of SEQ ID NO:1; particularly only 1, 2, 3, 4, 5 such substitutions or insertions (preferably substitutions), particularly only 1, 2, or 3 such substitutions or insertions (preferably substitutions), particularly only 1 or 2 such mutations (preferably substitutions), preferably only 1 such substitution or insertion (preferably substitution).

[0195] In the RSV-F protein of the present disclosure, the region corresponding to positions 345-352 of SEQ ID NO:1 (preferably comprising a beta-sheet and a loop) may have at least 50% or 60% sequence identity with positions 345-352 of SEQ ID NO:1, or preferably at least 75% or 85% sequence identity.

[0196] In some embodiments of the RSV-F protein of the present disclosure, one or more S residues (e.g., at positions 348 and / or 350) in the wild-type beta-sheet corresponding to positions 348-352 of SEQ ID NO:1 may be substituted with N, D, F, H, K, L, Q, R, T, W, or Y. For example, positions 348 and / or 350 of SEQ ID NO:1 may be substituted with N, F, H, K, N, Q, R, T, W, or Y, particularly N, F, R, W, or Y, preferably N.

[0197] In a preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 348 (S) of SEQ ID NO:1 with N, D, F, H, K, L, Q, R, T, W or Y. In a more preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 348 (S) of SEQ ID NO:1 with N, F, H, K, N, Q, R, T, W or Y. In a more preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 348 (S) of SEQ ID NO:1 with N, F, R, W or Y. In an even more preferred embodiment, the RSV-F protein of the present disclosure comprises a substitution at position 348 (S) of SEQ ID NO:1 with N. Such preferred substitutions at position 348 may be the only mutations according to (d); and optionally the only mutations in the region corresponding to positions 345 - 352 of SEQ ID NO:1.

[0198] As detailed in Example 6, the minimal substitution screen conducted by the inventors revealed that the S348N mutation may be a driver of the pre-fusion conformation (design F311). Additionally, alternative substitutions provided by the ROSETTA software for position 348 include: D, F, H, K, L, Q, R, T, W and Y (based on allowing all amino acids (no evolutionary constraints), using an energy threshold of 0.0 or -0.1), F, H, K, N, Q, R, T, W and Y (using the same parameters, except an energy threshold of -0.5), and F, R, W or Y (using the same parameters, except an energy threshold of -2).

[0199] Mutations according to (d) introducing an N residue or a T residue may introduce a glycosylation site into the above region (preferably comprising a β-sheet and a loop); preferably resulting in glycosylation of residues within the region, or alternatively defined as resulting in the introduction of a glycan linked to residues within the region. In such embodiments, the glycosylation site may be conserved by maintaining the wild-type residue (S) at position 350 or by replacing S350 with T.

[0200] In a specific embodiment, the RSV-F protein of the present disclosure further comprises at least one mutation (preferably a substitution) in the region corresponding to positions 416 - 422 of SEQ ID NO:1 (preferably forming a loop), wherein the at least one mutation increases the negative charge of the region (e.g., by introducing D and / or E residues). Preferably, the mutation is a substitution of D or E at position 419 of SEQ ID NO:1. In a more preferred embodiment, the mutation is a substitution of D at position 419 of SEQ ID NO:1. Such mutations (in the presence or absence of glycosylation) in the region corresponding to positions 416 - 422 of SEQ ID NO:1 (preferably forming a loop) can enhance the trans - protomer interaction, thereby contributing to the inhibition of the transition of RSV-F from the pre - fusion to the post - fusion conformation.

[0201] Generally, the mutation according to (d) (preferably such a substitution, preferably the substitution at position 348 as detailed above) can stabilize or rigidify the loop region corresponding to positions 346 - 347 of SEQ ID NO:1 in the F1 domain. This stabilization or rigidification can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre - fusion to the post - fusion conformation. Generally, the mutation according to (d) (preferably such a substitution, preferably the substitution at position 348 as detailed above) can provide trans - protomer interaction. This trans - protomer interaction can occur with one or more charged residues, including, for example, at position 419 of SEQ ID NO:1 (e.g., K, E, or D residues at position 419). This trans - protomer interaction can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre - fusion to the post - fusion conformation. Generally, the mutation according to (d) (preferably such a substitution, preferably the substitution at position 348 as detailed above) can inhibit, at least partially inhibit, or completely inhibit the transition of RSV-F from the pre - fusion to the post - fusion conformation.

[0202] (a), (b) and (c) or combination of (d)

[0203] In preferred embodiments according to all independent aspects of the present disclosure, the RSV-F protein of the present disclosure relative to SEQ ID NO:1 comprises:

[0204] (a) a substitution of S at position 55 of SEQ ID NO:1 with T, C, V, I, preferably T, C, or V, preferably T or V, more preferably T;

[0205] (b) a substitution of S at position 215 of SEQ ID NO:1 with A, P, V, I, or F, preferably A, V, I, or F, preferably A or P, more preferably A; and

[0206] (c) A substitution of S at position 348 of SEQ ID NO:1 with N or T, more preferably N; optionally, wherein the glycan is linked to said N or T at position 348.

[0207] The foregoing substitutions are preferably the only mutations relative to SEQ ID NO:1 according to (a), (b) and (c).

[0208] In a further preferred embodiment, the RSV-F protein of the present disclosure comprises, relative to SEQ ID NO:1:

[0209] (a) A substitution of S at position 55 of SEQ ID NO:1 with T, C, V, I, preferably T, C or V, preferably T or V, more preferably T;

[0210] (b) A substitution of S at position 215 of SEQ ID NO:1 with A, P, V, I or F, preferably A, V, I or F, preferably A or P, more preferably A; and

[0211] (d) A substitution of S at position 348 of SEQ ID NO:1 with N, D, F, H, K, L, Q, R, T, W or Y, preferably N, F, H, K, N, Q, R, T, W or Y, preferably N, F, R, W or Y, more preferably N.

[0212] The foregoing substitutions are preferably the only mutations relative to SEQ ID NO:1 according to (a), (b) and (d).

[0213] In a more preferred embodiment, the RSV-F protein of the present disclosure comprises the substitutions S55T, S215A and S348N relative to SEQ ID NO:1; optionally, wherein the glycan is linked to said N at position 348; optionally except for further mutations (preferably substitutions) as detailed below. The foregoing mutations are preferably the only mutations relative to SEQ ID NO:1 according to (a), (b) and ((c) or (d)).

[0214] Even further mutations

[0215] According to all independent aspects of the present disclosure, except for (a), (b) and / or ((c) or (d)) as detailed above, or vice versa, preferably in addition to them, the RSV-F protein of the present disclosure may comprise at least one further mutation, preferably at least one further substitution, relative to SEQ ID NO:1.

[0216] In some embodiments, the at least one further substitution is selected from (number and original residue according to SEQ ID NO:1):

[0217] A74R; substitution at position 152 (V) with R, L, or W; S169E; S180E; S190I; substitution at position 210 (Q) with H, A, F, K, N, W, or Y; S211N; E218; K226L; substitution at position 228 (N) with K, R, or A; A241N; M251L; S275L; M289L; V296I; L305I; substitution at position 315 (K) with I or V; T326D, substitution at position 346 (A) with Q, D, H, K, N, R, S, or W; S350I, K359I; V384K; substitution at position 419 (K) with D, N, S, or T; K445D; substitution at position 455 (T) with V or I; V459M; F477R; E487Q and Q501K. Except for (a), (b), and / or ((c) or (d)), the RSV-F protein of the present disclosure may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or at least 50 of the foregoing substitutions; such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 of the foregoing substitutions. In a specific embodiment, except for (a), (b), and / or ((c) or (d)), the RSV-F protein of the present disclosure comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 of the foregoing substitutions, such as no more than 14, such as no more than 11, such as no more than 8, such as no more than 5, such as no more than 4 of the foregoing substitutions.

[0218] In a specific embodiment, the substitutions V152R and / or A346Q may be present in the RSV-F protein of the present disclosure. Such substitutions may enhance the expression of the RSV-F protein. V152R and A346Q are surface-exposed substitutions present in two designs, F216 and F217, which show higher levels of in vitro expression from mRNA than F224 and F225 (only buried substitutions), see, for example, Example 7; Figure 21 .

[0219] In a specific embodiment, the substitutions S211N and / or K445D (in particular, both) may be present in the RSV-F protein of the present disclosure. As illustrated in Example 7, the presence of S211N and K445D is shown to improve the stability of the protein after heat stress (see Figure 30, comparing F217 (with both substitutions) with F318 (lacking S211N), and comparing F216 (with both substitutions) with F319 (lacking both substitutions)).

[0220] In a preferred embodiment, in addition to (a), (b) and / or ((c) or (d)), or alternatively thereto, preferably in addition to these, the RSV-F protein of the present disclosure may comprise at least 1, such as at least 2, 3, 4, 5, 6, 7, 8, 9 or at least 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11) substitutions selected from the following (numbering and original residues according to SEQ ID NO: 1):

[0221] A substitution at position 152 (V) with R, L or W, preferably R or W, preferably R;

[0222] A substitution at position 210 (Q) with H, A, F, K, N, W or Y, preferably H, F, K, N, W or Y, preferably H, F or Y; preferably H;

[0223] Optionally, a substitution at position 211 (S) with N;

[0224] A substitution at position 241 (A) with N

[0225] A substitution at position 315 (K) with I or V, preferably I;

[0226] A substitution at position 346 (A) with Q, D, H, K, N, R, S or W, preferably Q, D, H, K, N, R or S, preferably Q;

[0227] A substitution at position 419 (K) with D, N, S or T, preferably D or T, preferably D;

[0228] Optionally, a substitution at position 445 (K) with D;

[0229] A substitution at position 455 (T) with V or I, preferably V; and

[0230] A substitution at position 459 (V) with M.

[0231] Except for alternative residues provided by ROSETTA software (which allows all amino acids (no evolutionary constraints) with an energy threshold of 0.0) and / or visual analysis of three-dimensional structures, the foregoing substitutions at positions 152, 210, 211, 241, 315, 346, 419, 445, 455, and 459 include those present in Design F216 (F216 also has S55T, S215A, N228K, and S348N); a more stringent ROSETTA energy threshold and / or visual analysis were used to generate the subset. In a more preferred embodiment, the RSV-F protein of the present disclosure comprises the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V, and V459M relative to SEQ ID NO:1, and optionally S211N and / or K445D (all of these substitutions are present in Design F216, see, for example, Example 4), optionally wherein the glycan is N-linked at position 316; optionally there are no further mutations relative to SEQ ID NO:1.

[0232] In a preferred embodiment, in addition to (a), (b), and / or ((c) or (d)), or in contrast thereto, preferably in addition to them, the RSV-F protein of the present disclosure may comprise at least 1, such as at least 2, 3, 4, 5, 6, or at least 7 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) substitutions selected from the following (numbering and original residues according to SEQ ID NO:1):

[0233] A substitution of V at position 152 with R, L, or W, preferably R or W, preferably R;

[0234] Optionally, a substitution of S at position 211 with N;

[0235] A substitution of K at position 315 with I or V, preferably I;

[0236] A substitution of A at position 346 with Q, D, H, K, N, R, S, or W, preferably Q, D, H, K, N, R, or S, preferably Q;

[0237] Optionally, a substitution of K at position 445 with D;

[0238] A substitution of T at position 455 with V or I, preferably V; and

[0239] A substitution of V at position 459 with M.

[0240] Except for alternative residues suggested by ROSETTA software (based on allowing all amino acids (no evolutionary constraints), using an energy threshold of 0.0) and / or visual analysis of three-dimensional structures, the foregoing substitutions at positions 152, 211, 315, 346, 445, 455, and 459 include the substitutions present in Design F217 (F217 also has S55T, S215A, N228K, and S348N); a more stringent ROSETTA energy threshold and / or visual analysis were used to generate the subset. In a more preferred embodiment, the RSV-F protein of the present disclosure comprises the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, T455V, and V459M relative to SEQ ID NO:1, and optionally S211N and / or K445D (e.g., as present in Design F217, see, e.g., Example 4); optionally wherein the glycan is N-linked at position 348; and optionally there are no further mutations relative to SEQ ID NO:1.

[0241] In a preferred embodiment, in addition to (a), (b), and / or ((c) or (d)), or alternatively thereto, preferably in addition to these, the RSV-F protein of the present disclosure may comprise at least 1, such as at least 2, 3, or at least 4 (e.g., 1, 2, 3, 4, or 5) substitutions selected from the following (numbering and original residues according to SEQ ID NO:1):

[0242] A substitution of K at position 315 with I or V, preferably I;

[0243] A substitution of A at position 241 with N

[0244] A substitution of T at position 455 with V or I, preferably V; and

[0245] A substitution of V at position 459 with M.

[0246] Except for alternative residues provided by ROSETTA software (based on allowing all amino acids (no evolutionary constraints), using an energy threshold of 0.0) and / or visual analysis of the three-dimensional structure, the foregoing substitutions at positions 241, 315, 455, and 459 include the substitutions present in Design F224 (F224 also has S55T, S215A, N228K, and S348N); a more stringent ROSETTA energy threshold and / or visual analysis were used to generate the subset. In a more preferred embodiment, the RSV-F protein of the present disclosure comprises the substitutions S55T, S215A, N228K, A241N, K315I, S348N, T455V, and V459M relative to SEQ ID NO:1 (e.g., as present in Design F224, see, e.g., Example 4); optionally wherein the glycan is N-linked at position 348; optionally there are no further mutations relative to SEQ ID NO:1.

[0247] In a preferred embodiment, in addition to (a), (b), and / or ((c) or (d)), or vice versa, preferably in addition thereto, the RSV-F protein of the present disclosure may comprise at least 1, such as at least 2, or at least 3 (e.g., 1, 2, 3, or 4) substitutions selected from the following (numbering and original residues according to SEQ ID NO:1):

[0248] A substitution at position 315 (K) with I or V, preferably I;

[0249] A substitution at position 455 (T) with V or I, preferably V; and

[0250] A substitution at position 459 (V) with M.

[0251] Except for alternative residues provided by ROSETTA software (based on allowing all amino acids (no evolutionary constraints), using an energy threshold of 0.0) and / or visual analysis of the three-dimensional structure, the foregoing substitutions at positions 315, 455, and 459 include the substitutions present in Design F225 (F225 also has S55T, S215A, N228K, and S348N); a more stringent ROSETTA energy threshold and / or visual analysis were used to generate the subset. In a more preferred embodiment, the RSV-F protein of the present disclosure comprises the substitutions S55T, S215A, N228K, K315I, S348N, T455V, and V459M relative to SEQ ID NO:1 (as present in Design F225, see, e.g., Example 4); optionally wherein the glycan is N-linked at position 348; optionally there are no further mutations relative to SEQ ID NO:1.

[0252] In general, further mutations as detailed above in this subsection (preferably such substitutions, preferably such substitutions at positions 152, 210, 211, 241, 315, 346, 419, 445, 455 and / or 459 as detailed above) can inhibit, at least partially inhibit or completely inhibit the transition of RSV-F from the pre-fusion to the post-fusion conformation.

[0253] General sequence characteristics of RSV-F protein (the protein itself) in pre-fusion conformation

[0254] When considering the protein itself (e.g., the mature furin-processed protein), the RSV-F protein of the present disclosure generally has two domains (in the N-terminal to C-terminal direction, the "F2" domain and the "F1" domain), which may or may not be connected by a peptide bond (although in the wild-type protein they are not so connected; the connection usually occurs through a disulfide bond). The F2 domain may have at least 70% sequence identity with positions 26-108 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with positions 26-108; and the F1 domain may have at least 70% sequence identity with positions 137-513 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with positions 137-513 of SEQ ID NO:1. The F2 domain may have at least 70% sequence identity with positions 26-109 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98% or 99% sequence identity with positions 26-109; and the F1 domain may have at least 70% sequence identity with positions 137-513 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.2% or 99.5% sequence identity with positions 137-513 of SEQ ID NO:1.

[0255] In a preferred embodiment, the signal peptide is absent from the RSV-F protein of the present disclosure, optionally as a result of signal peptide cleavage, optionally wherein the signal peptide is positions 1-25 of SEQ ID NO:1.

[0256] In some embodiments, the RSV-F protein of the present disclosure comprises an E residue at position 66 of SEQ ID NO:1 and a P residue at position 101.

[0257] The RSV-F protein of the present disclosure can have at least 70% sequence identity with SEQ ID NO:13, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with SEQ ID NO:13. The RSV-F protein of the present disclosure can have at least 70% sequence identity with SEQ ID NO:84, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with SEQ ID NO:84. SEQ ID NO:13 and 84 are the mature furin-processed sequences of wild-type RSV-F from subtype A2 (i.e., SEQ ID NO:1 without the signal sequence and p27).

[0258] In the embodiments of the previous paragraph and in all embodiments presented below in this subsection, positions 84 (R) and 85 (F) of SEQ ID NOs:13, 28 - 38, 50 - 59, and 84 - 106 (positions 109 and 137 of SEQ ID NO:1, respectively) are generally discontinuous and may or may not (preferably not) be connected by an intervening amino acid sequence (such as a linker sequence). That is, positions 1 - 84 of SEQ ID NOs:13, 28 - 38, 50 - 59, and 84 - 106 form (in whole or in part) the F2 domain, and positions 85 - 461 of said sequences form (in whole or in part) the F1 domain, where the F2 domain and the F1 domain may or may not (preferably not) be connected by an intervening amino acid sequence (such as a linker sequence) between positions 84 and 85 of said sequences. In the mature furin-processed protein, the p27 peptide may still be present as a result of furin cleavage occurring at only one site, for example, the p27 peptide is connected to one of the F2 domain or the F1 domain by a peptide bond.

[0259] In a preferred embodiment, the RSV-F protein of the present disclosure comprises or consists of the amino acid sequence according to SEQ ID NO: 28 or 85, or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain as compared to its full-length sequence). The portion preferably contains the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 28 and 85.

[0260] In a preferred embodiment, the RSV-F protein of the present disclosure comprises or consists of the amino acid sequence according to SEQ ID NO: 29 or 86, or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain as compared to its full-length sequence). The portion preferably contains the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 29 and 86.

[0261] In a preferred embodiment, the RSV-F protein of the present disclosure comprises or consists of the amino acid sequence according to SEQ ID NO: 30 or 87, or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain as compared to its full-length sequence). The portion preferably contains the substitutions S55T, S215A, N228K, A241N, K315I, S348N, T455V and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 30 and 87.

[0262] In a preferred embodiment, the RSV-F protein of the present disclosure comprises, consists of, or consists essentially of the amino acid sequence according to SEQ ID NO: 31 or 88, or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length). The portion preferably encompasses all or part of the F2 domain and the F1 domain (i.e., at least part of the domain compared to its full-length sequence). The portion preferably contains the substitutions S55T, S215A, N228K, K315I, S348N, T455V, and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 31 and 88.

[0263] In a preferred embodiment, the RSV-F protein of the present disclosure comprises, consists of, or consists essentially of the amino acid sequence according to SEQ ID NO: 33 or 90, or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least part of the domain compared to its full-length sequence). The portion preferably contains the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V, and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 33 and 90.

[0264] In a preferred embodiment, the RSV-F protein of the present disclosure comprises, consists of, or consists essentially of the amino acid sequence according to SEQ ID NO: 34 or 91, or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least part of the domain compared to its full-length sequence). The portion preferably contains the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V, and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 34 and 91.

[0265] In a preferred embodiment, the RSV-F protein of the present disclosure comprises, consists of, or consists essentially of the amino acid sequence according to SEQ ID NO: 35 or 92 or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain as compared to its full-length sequence). The portion preferably contains the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V, and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 35 and 92.

[0266] In a preferred embodiment, the RSV-F protein of the present disclosure comprises, consists of, or consists essentially of the amino acid sequence according to SEQ ID NO: 36 or 93 or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain as compared to its full-length sequence). The portion preferably contains the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, T455V, and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 36 and 93.

[0267] In a preferred embodiment, the RSV-F protein of the present disclosure comprises, consists of, or consists essentially of the amino acid sequence according to SEQ ID NO: 37 or 94 or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain as compared to its full-length sequence). The portion preferably contains the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V, and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 37 and 94.

[0268] In a preferred embodiment, the RSV-F protein of the present disclosure comprises, consists of, or consists essentially of the amino acid sequence according to SEQ ID NO: 38 or 95, or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain as compared to its full-length sequence). The portion preferably comprises the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, T455V, and V459M (numbering according to SEQ ID NO: 1), which are present in SEQ ID NO: 38 and 95.

[0269] In a preferred embodiment, the RSV-F protein of the present disclosure comprises, consists of, or consists essentially of the amino acid sequence according to SEQ ID NO: 82 or 106, or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain as compared to its full-length sequence). The portion comprises the substitution N228K (numbering according to SEQ ID NO: 1), which is present in SEQ ID NO: 82 and 106.

[0270] In a further embodiment, the RSV-F protein of the present disclosure can comprise, consist of, or consist essentially of the amino acid sequence according to any one of SEQ ID NOs: 50-53, 55, or 56, or a portion of any of the foregoing (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain as compared to its full-length sequence). The portion preferably comprises all substitutions relative to SEQ ID NO: 13 that are present in the amino acid sequence according to any one of SEQ ID NOs: 50-53, 55, 56 (as applicable).

[0271] In a further embodiment, the RSV-F protein of the present disclosure can comprise, consist of, or consist essentially of the amino acid sequence according to any one of SEQ ID NOs: 96-99, 101, or 102, or a portion of any of the foregoing (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99%, or 99.5% of its length). The portion preferably encompasses the F2 domain and the F1 domain (i.e., at least a portion of the domain as compared to its full-length sequence). The portion preferably comprises all substitutions relative to SEQ ID NO: 84 that are present in the amino acid sequence according to any one of SEQ ID NOs: 96-99, 101, or 102 (as applicable).

[0272] In embodiments in which the F2 domain and the F1 domain are linked by peptide bonds (such as those of the intervening amino acid sequence), they may be linked by a linker sequence. The linker sequence will link the C-terminal and N-terminal regions / residues of the F2 domain and the F1 domain. The linker sequence may be glycine-serine rich or consist of G and S residues, such as GSGSG (SEQ ID NO:10), GSGSGRS (SEQ ID NO:11) or GS (SEQ ID NO:12). In a specific embodiment, the "F2" domain and the "F1" domain may be linked by a linker comprising or consisting of SEQ ID NO:11 (or a linker having at least 55%, 75% or 85% identity thereto). In an alternative specific embodiment, the "F2" domain and the "F1" domain may be linked by a linker comprising or consisting of SEQ ID NO:12 (or G or S residues). In embodiments in which the "F2" domain and the "F1" domain are not linked by peptide bonds, they may be linked by at least one disulfide bond (usually two such bonds, which are usually naturally occurring, such as in the wild-type protein).

[0273] The RSV-F protein of the present disclosure may have at least 70% sequence identity with SEQ ID NO:13 over at least 80% of SEQ ID NO:13; in particular, it has at least 75% sequence identity with SEQ ID NO:13 over at least 80% of SEQ ID NO:13, at least 80% sequence identity with SEQ ID NO:13 over at least 80% of SEQ ID NO:13, at least 85% sequence identity with SEQ ID NO:13 over at least 80% of SEQ ID NO:13, at least 90% sequence identity with SEQ ID NO:13 over at least 80% of SEQ ID NO:13, at least 95% sequence identity with SEQ ID NO:13 over at least 80% of SEQ ID NO:13, at least 99% sequence identity with SEQ ID NO:13 over at least 80% of SEQ ID NO:13, at least 99.4% sequence identity with SEQ ID NO:13 over at least 80% of SEQ ID NO:13, at least 99.5% sequence identity with SEQ ID NO:13 over at least 80% of SEQ ID NO:13, at least 75% sequence identity with SEQ ID NO:13 over at least 90% of SEQ ID NO:13, at least 80% sequence identity with SEQ ID NO:13 over at least 90% of SEQ ID NO:13, at least 85% sequence identity with SEQ ID NO:13 over at least 90% of SEQ ID NO:13, at least 90% sequence identity with SEQ ID NO:13 over at least 90% of SEQ ID NO:13, at least 95% sequence identity with SEQ ID NO:13 over at least 90% of SEQ ID NO:13, at least 99% sequence identity with SEQ ID NO:13 over at least 90% of SEQ ID NO:13, at least 99.4% sequence identity with SEQ ID NO:13 over at least 90% of SEQ ID NO:13, at least 99.5% sequence identity, having at least 75% sequence identity with SEQ ID NO:13 over at least 95% of SEQ ID NO:13, having at least 80% sequence identity with SEQ ID NO:13 over at least 95% of SEQ ID NO:13, having at least 85% sequence identity with SEQ ID NO:13 over at least 95% of SEQ ID NO:13, having at least 90% sequence identity with SEQ ID NO:13 over at least 95% of SEQ ID NO:13, having at least 95% sequence identity with SEQ ID NO:13 over at least 95% of SEQ ID NO:13, having at least 99% sequence identity with SEQ ID NO:13 over at least 95% of SEQ ID NO:13, having at least 99.4% sequence identity with SEQ ID NO:13 over at least 95% of SEQ ID NO:13, or having at least 99.5% sequence identity with SEQ ID NO:13 over at least 95% of SEQ ID NO:13..

[0274] The RSV-F protein of the present disclosure can have at least 70% sequence identity with SEQ ID NO:13, such as having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with SEQ ID NO:13 over 100% of SEQ ID NO 13.

[0275] The RSV-F protein of the present disclosure can have at least 70% sequence identity with SEQ ID NO:84 over at least 80% of SEQ ID NO:84; in particular, it can have at least 75% sequence identity with SEQ ID NO:84 over at least 80% of SEQ ID NO:84, at least 80% sequence identity with SEQ ID NO:84 over at least 80% of SEQ ID NO:84, at least 85% sequence identity with SEQ ID NO:84 over at least 80% of SEQ ID NO:84, at least 90% sequence identity with SEQ ID NO:84 over at least 80% of SEQ ID NO:84, at least 95% sequence identity with SEQ ID NO:84 over at least 80% of SEQ ID NO:84, at least 99% sequence identity with SEQ ID NO:84 over at least 80% of SEQ ID NO:84, at least 99.4% sequence identity with SEQ ID NO:84 over at least 80% of SEQ ID NO:84, at least 99.5% sequence identity with SEQ ID NO:84 over at least 80% of SEQ ID NO:84, at least 75% sequence identity with SEQ ID NO:84 over at least 90% of SEQ ID NO:84, at least 80% sequence identity with SEQ ID NO:84 over at least 90% of SEQ ID NO:84, at least 85% sequence identity with SEQ ID NO:84 over at least 90% of SEQ ID NO:84, at least 90% sequence identity with SEQ ID NO:84 over at least 90% of SEQ ID NO:84, at least 95% sequence identity with SEQ ID NO:84 over at least 90% of SEQ ID NO:84, at least 99% sequence identity with SEQ ID NO:84 over at least 90% of SEQ ID NO:84, at least 99.4% sequence identity with SEQ ID NO:84 over at least 90% of SEQ ID NO:84, at least 99.5% sequence identity, having at least 75% sequence identity with SEQ ID NO:84 over at least 95% of SEQ ID NO:84, having at least 80% sequence identity with SEQ ID NO:84 over at least 95% of SEQ ID NO:84, having at least 85% sequence identity with SEQ ID NO:84 over at least 95% of SEQ ID NO:84, having at least 90% sequence identity with SEQ ID NO:84 over at least 95% of SEQ ID NO:84, having at least 95% sequence identity with SEQ ID NO:84 over at least 95% of SEQ ID NO:84, having at least 99% sequence identity with SEQ ID NO:84 over at least 95% of SEQ ID NO:84, having at least 99.4% sequence identity with SEQ ID NO:84 over at least 95% of SEQ ID NO:84, or having at least 99.5% sequence identity with SEQ ID NO:84 over at least 95% of SEQ ID NO:84..

[0276] The RSV-F protein of the present disclosure can have at least 70% sequence identity with SEQ ID NO:84, such as having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with SEQ ID NO:84 over 100% of SEQ ID NO 84.

[0277] When considering the protein itself, in a preferred embodiment, the RSV-F protein of the present disclosure comprises a heterotrimerization domain at its C-terminus ("hetero" indicating not native to the viral protein). Additionally, or alternatively, the trimerization domain may be located at the C-terminus of the F1 domain. The trimerization domain is a sequence that promotes the assembly of the RSV-F protein of the present disclosure (i.e., a single protomer) into a trimer, i.e., specifically by associating with other trimerization domains (i.e., those on other protomers). In some embodiments, the trimerization domain may fold into a coiled coil. Exemplary trimerization domains include: the T4 minor fibritin foldon domain; the yeast GCN4 leucine zipper, e.g., according to SEQ ID NO:39 (or an amino acid sequence that is at least 50%, 60%, 70%, 80%, 90% or 95% identical thereto, particularly having trimerization function); TRAF2 (GENBANK accession number Q12933 [gi:23503103]; amino acids 299 - 348); thrombospondin 1 (accession number PO7996 [gi:135717]; amino acids 291 - 314); Matrilin-4 (accession number 095460 [gi:14548117]; amino acids 594 - 618); CMP (matrilin-1) (accession number NP_002370 [gi:4505111]; amino acids 463 - 496); HSF1 (accession number AAX42211 [gi:61362386]; amino acids 165 - 191); Cubilin (accession number NP_001072 [gi:4557503]; amino acids 104 - 138); the trimerization domain from influenza hemagglutinin; the trimerization domain from the SARS spike protein, the trimerization domain from HIV gp41; NadA; and aspartate transcarbamoylase (ATCase). Preferably, the trimerization domain is the T4 minor fibritin foldon domain, more preferably comprising (or consisting of) the amino acid sequence according to SEQ ID NO:14 (or an amino acid sequence that is at least 50%, 60%, 70%, 80%, 90% or 95% identical thereto, preferably having trimerization function). The trimerization domain is preferably linked to the C-terminus (i.e., the F1 domain) of the RSV-F protein of the present disclosure via a linker sequence. The linker sequence preferably comprises (or consists of) the amino acid sequence according to SEQ ID NO:60 (or an amino acid sequence that is at least 50% or 75% identical thereto).

[0278] As noted above, a fourth independent aspect of the present disclosure is a multimer that includes protomers, where at least one protomer is the RSV-F protein of the present disclosure. Preferably, the multimer is a trimer of the RSV-F protein of the present disclosure. Preferably, the trimer is a homotrimer (i.e., includes three RSV-F proteins of the present disclosure that include or are composed of the same primary amino acid sequence).

[0279] Preparation of RSV-F protein in pre-fusion conformation

[0280] The RSV-F protein of the present disclosure can be prepared by conventional methods, such as by expression in a recombinant host system using a nucleic acid expression vector (e.g., an expression vector as detailed in the section titled "Nucleic Acids Encoding the RSV-F Protein" below).

[0281] Suitable recombinant host cells include, for example, insect cells (e.g., Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells); mammalian cells (e.g., Chinese hamster ovary (CHO) cells, human embryonic kidney cells (e.g., HEK293, particularly Expi 293 cells), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells); avian cells (e.g., chicken embryo fibroblasts and chicken embryo germ cells); bacteria; and yeast cells. HEK293 cells are preferred, and Expi 293 cells (as used in the examples) are more preferred. Thus, in an independent aspect, the present disclosure also provides a host cell (particularly, those host cells detailed above) that includes a nucleic acid encoding the RSV-F protein of the present disclosure (particularly, an expression vector as detailed below). In a further independent aspect, the present disclosure also provides a host cell (particularly, those host cells detailed above) that includes and / or expresses the RSV-F protein of the present disclosure. In a further independent aspect, the present invention also provides a composition that includes a host cell (particularly, those host cells detailed above) and (i) a nucleic acid encoding the RSV-F protein of the present disclosure (particularly, an expression vector as detailed below), and / or (ii) the RSV-F protein of the present disclosure. In a further independent aspect, the present disclosure also provides an in vitro method for producing the RSV-F protein of the present disclosure, which includes expressing a nucleic acid encoding the RSV-F protein (particularly, an expression vector as detailed below) in a host cell (particularly, those host cells detailed above), and optionally purifying the RSV-F protein.

[0282] The RSV-F protein of the present disclosure can be purified by conventional methods after expression from host cells, such as precipitation and chromatography methods (e.g., hydrophobic interaction, ion exchange, affinity, chelation, or size exclusion chromatography). The RSV-F protein of the present disclosure can contain tags that facilitate purification, such as epitope tags or histidine (HIS) tags, to facilitate purification by, for example, affinity chromatography.

[0283] Nucleic acid encoding RSV-F protein in pre-fusion conformation

[0284] In a further independent aspect, the present disclosure also provides a nucleic acid encoding the RSV-F protein of the present disclosure.

[0285] General sequence characteristics of RSV-F protein in pre-fusion conformation when encoded by nucleic acid (such as RNA)

[0286] The nucleic acid of the present disclosure can encode an RSV-F protein of the present disclosure that has at least 70% sequence identity with SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% sequence identity with SEQ ID NO:1. SEQ ID NO:1 is the sequence of wild-type RSV-F from subtype A2, which contains a signal sequence (positions 1-25 of SEQ ID NO:1), and a p27 peptide (positions 109-136 or 110-136 of SEQ ID NO:1), which is cleaved by furin processing in the mature protein.

[0287] The nucleic acid of the present disclosure can encode an RSV-F protein of the present disclosure that contains an F2 domain having at least 70% sequence identity with positions 26-109 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity with positions 26-109 of SEQ ID NO:1; and an F1 domain having at least 70% sequence identity with positions 137-513 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.2%, or 99.5% sequence identity with positions 137-513 of SEQ ID NO:1

[0288] In some embodiments, the RSV-F protein of the present disclosure comprises an E residue at position 66 of SEQ ID NO:1 and a P residue at position 101.

[0289] In a specific embodiment, the signal peptide (positions 1-25 of SEQ ID NO:1) is not considered in the above sequence identity assessment. Thus, in some embodiments, the nucleic acid of the present disclosure encodes the RSV-F protein of the present disclosure, which has at least 70% sequence identity with SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% identity with positions 26-513 of SEQ ID NO:1.

[0290] In a preferred embodiment, the nucleic acid of the present disclosure encodes an RSV-F protein comprising the amino acid sequence according to SEQ ID NO:17; or a portion thereof (such as at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consisting of the RSV-F protein. The portion preferably comprises the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M present in SEQ ID NO:17.

[0291] In a preferred embodiment, the nucleic acid of the present disclosure encodes an RSV-F protein comprising the amino acid sequence according to SEQ ID NO:18; or a portion thereof (such as at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consisting of the RSV-F protein. The portion preferably comprises the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M present in SEQ ID NO:18.

[0292] In a preferred embodiment, the nucleic acid encoding of the present disclosure comprises an amino acid sequence according to SEQ ID NO: 19; or a portion thereof (such as at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or an RSV-F protein consisting of the same. The portion preferably comprises the substitutions S55T, S215A, N228K, A241N, K315I, S348N, T455V and V459M present in SEQ ID NO: 19.

[0293] In a preferred embodiment, the nucleic acid encoding of the present disclosure comprises an amino acid sequence according to SEQ ID NO: 20; or a portion thereof (such as at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or an RSV-F protein consisting of the same. The portion preferably comprises the substitutions S55T, S215A, N228K, K315I, S348N, T455V and V459M present in SEQ ID NO: 20.

[0294] In a preferred embodiment, the nucleic acid encoding of the present disclosure comprises an amino acid sequence according to SEQ ID NO: 22 or a portion thereof (such as at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or an RSV-F protein consisting of the same. The portion preferably comprises the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M present in SEQ ID NO: 22.

[0295] In a preferred embodiment, the nucleic acid encoding of the present disclosure comprises an amino acid sequence according to SEQ ID NO: 23 or a portion thereof (such as at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or an RSV-F protein consisting of the same. The portion preferably comprises the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M present in SEQ ID NO: 23.

[0296] In a preferred embodiment, the nucleic acid encoding of the present disclosure comprises an RSV-F protein comprising the amino acid sequence according to SEQ ID NO: 24 or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consisting of the same. The portion preferably comprises the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M present in SEQ ID NO: 24.

[0297] In a preferred embodiment, the nucleic acid encoding of the present disclosure comprises an RSV-F protein comprising the amino acid sequence according to SEQ ID NO: 25 or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consisting of the same. The portion preferably comprises the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, T455V and V459M present in SEQ ID NO: 25.

[0298] In a preferred embodiment, the nucleic acid encoding of the present disclosure comprises an RSV-F protein comprising the amino acid sequence according to SEQ ID NO: 26 or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consisting of the same. The portion preferably comprises the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M present in SEQ ID NO: 26.

[0299] In a preferred embodiment, the nucleic acid encoding of the present disclosure comprises an RSV-F protein comprising the amino acid sequence according to SEQ ID NO: 27 or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consisting of the same. The portion preferably comprises the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, T455V and V459M present in SEQ ID NO: 27.

[0300] In a preferred embodiment, the nucleic acid encoding of the present disclosure comprises an RSV-F protein comprising the amino acid sequence according to SEQ ID NO: 81 or a portion thereof (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consisting of the same. The portion preferably comprises the substitution N228K present in SEQ ID NO: 81.

[0301] In a further embodiment, the nucleic acid of the present disclosure can encode an RSV-F protein comprising the amino acid sequence according to any one of SEQ ID NOs: 40-43, 45 or 46 or a portion of any one of the foregoing (such as a portion that is at least 70%, 80%, 85%, 90%, 95%, 99% or 99.5% of its length), or consisting of the same. The portion preferably comprises all substitutions relative to SEQ ID NO: 1 present in the amino acid sequence according to any one of SEQ ID NOs: 40-43, 45 or 46 (if applicable).

[0302] Two furin cleavage sites are present between positions 108 and 137 of SEQ ID NO: 1 (positions 109-136 or 110-136 of SEQ ID NO: 1 that define the "p27" peptide). In some embodiments, the nucleic acid of the present disclosure encodes the RSV-F protein of the present disclosure, wherein the p27 peptide is artificially absent (i.e., at the level of the encoding nucleic acid, there is an artificial absence of the p27 peptide, such as by recombinant means). In such embodiments, the fusion peptide (positions 137-157 of SEQ ID NO: 1) may also be artificially absent. In some embodiments, the p27 peptide (and optionally, the fusion peptide) can be replaced with a linker sequence encoded by the nucleic acid. The linker sequence can be glycine-serine rich (or consisting of G residues and S residues), such as GSGSG (SEQ ID NO: 10), GSGSGRS (SEQ ID NO: 11) or GS (SEQ ID NO: 12). In a specific embodiment, the p27 peptide (or at least 80%, 85%, 90% or 95% of its residues) is artificially absent and replaced with a linker comprising SEQ ID NO: 11 or consisting of the same (or a linker having at least 55%, 75% or 85% identity thereto). In an alternative specific embodiment, both the p27 peptide and the fusion peptide (or at least 80%, 85%, 90% or 95% of their residues) are artificially absent and replaced with a linker comprising SEQ ID NO: 12 (or G or S residues) or consisting of the same.

[0303] In embodiments where the p27 peptide is absent (including embodiments where the fusion peptide is also absent), the nucleic acids of the present disclosure can encode an RSV-F protein of the present disclosure comprising two domains (in the N-terminal to C-terminal direction, the "F2" domain and the "F1" domain); the F2 domain has at least 70% sequence identity with positions 1-108 or 1-109 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with positions 1-108 or 1-109 of SEQ ID NO:1; and the F1 domain has at least 70% sequence identity with positions 137-513 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with positions 137-513 of SEQ ID NO:1.

[0304] Alternatively, in embodiments where the p27 peptide is absent (including embodiments where the fusion peptide is also absent), the nucleic acids of the present disclosure can encode an RSV-F protein of the present disclosure comprising two domains (in the N-terminal to C-terminal direction, the "F2" domain and the "F1" domain); the F2 domain has at least 70% sequence identity with positions 26-108 or 26-109 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with positions 26-108 or 26-109; and the F1 domain has at least 70% sequence identity with positions 137-513 of SEQ ID NO:1, such as at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with positions 137-513 of SEQ ID NO:1.

[0305] The nucleic acids of the present disclosure can also encode the RSV-F protein of the present disclosure, which has at least 70% sequence identity with SEQ ID NO:1 over at least 80% of SEQ ID NO:1; in particular, having at least 75% sequence identity with SEQ ID NO:1 over at least 80% of SEQ ID NO:1, having at least 80% sequence identity with SEQ ID NO:1 over at least 80% of SEQ ID NO:1, having at least 85% sequence identity with SEQ ID NO:1 over at least 80% of SEQ ID NO:1, having at least 90% sequence identity with SEQ ID NO:1 over at least 80% of SEQ ID NO:1, having at least 95% sequence identity with SEQ ID NO:1 over at least 80% of SEQ ID NO:1, having at least 99% sequence identity with SEQ ID NO:1 over at least 80% of SEQ ID NO:1, having at least 99.4% sequence identity with SEQ ID NO:1 over at least 80% of SEQ ID NO:1, having at least 99.5% sequence identity with SEQ ID NO:1 over at least 80% of SEQ ID NO:1, having at least 75% sequence identity with SEQ ID NO:1 over at least 90% of SEQ ID NO:1, having at least 80% sequence identity with SEQ ID NO:1 over at least 90% of SEQ ID NO:1, having at least 85% sequence identity with SEQ ID NO:1 over at least 90% of SEQ ID NO:1, having at least 90% sequence identity with SEQ ID NO:1 over at least 90% of SEQ ID NO:1, having at least 95% sequence identity with SEQ ID NO:1 over at least 90% of SEQ ID NO:1, having at least 99% sequence identity with SEQ ID NO:1 over at least 90% of SEQ ID NO:1, having at least 99.4% sequence identity with SEQ ID NO:1 over at least 90% of SEQ ID NO:1, having at least 99.5% sequence identity, having at least 75% sequence identity with SEQ ID NO:1 over at least 95% of SEQ ID NO:1, having at least 80% sequence identity with SEQ ID NO:1 over at least 95% of SEQ ID NO:1, having at least 85% sequence identity with SEQ ID NO:1 over at least 95% of SEQ ID NO:1, having at least 90% sequence identity with SEQ ID NO:1 over at least 95% of SEQ ID NO:1, having at least 95% sequence identity with SEQ ID NO:1 over at least 95% of SEQ ID NO:1, having at least 99% sequence identity with SEQ ID NO:1 over at least 95% of SEQ ID NO:1, having at least 99.4% sequence identity with SEQ ID NO:1 over at least 95% of SEQ ID NO:1, or having at least 99.5% sequence identity with SEQ ID NO:1 over at least 95% of SEQ ID NO:1.

[0306] The nucleic acids of the present disclosure preferably encode an RSV-F protein of the present disclosure having at least 70% sequence identity with SEQ ID NO:1, such as having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or preferably at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with SEQ ID NO:1 over 100% of SEQ ID NO 1.

[0307] The nucleic acids of the present disclosure preferably encode an RSV-F protein comprising a transmembrane domain and optionally a cytoplasmic domain connected (directly or indirectly) to its C-terminus at the C-terminus of the transmembrane domain (i.e., at the C-terminus of position 513 of SEQ ID NO:1, or differently defined as at the C-terminus of the F1 domain). In some embodiments, there is no complete cytoplasmic domain. Preferably, the transmembrane domain comprises (or consists of) the amino acid sequence according to SEQ ID NO:15 (or a sequence having at least 80%, 85%, 90% or 95% identity thereto). Preferably, the cytoplasmic domain (if present) comprises (or consists of) the amino acid sequence according to SEQ IDNO:16, 109 or 110 (or a sequence having at least 80%, 85%, 90%, 95% or 95% identity thereto).

[0308] Nucleic acid encoding RSV-F protein with cytoplasmic tail deletion

[0309] The terms "cytoplasmic domain" and "cytoplasmic tail" are used interchangeably herein (including in the appended numbered embodiments and claims).

[0310] When expressed from nucleic acid in vitro, cell surface expression of the pre-fusion RSV-F trimer has been enhanced by deleting residues from the C-terminal cytoplasmic tail (see, e.g., Example 12). Additionally, surprisingly, deletion of 15, 16, 17, and 20 C-terminal residues resulted in higher pre-fusion RSV-F trimer expression at 72 and 96 hours post-transfection compared to deletion of 21 C-terminal residues (see, e.g., Example 14; Figure 45A ). Further, in vivo, at the lower of two different nucleic acid doses tested, RSV-F constructs comprising a cytoplasmic tail deletion generally elicited higher neutralizing antibody titers against, e.g., RSV of subtype A compared to their counterparts with a fully intact cytoplasmic tail (see, e.g., Example 13; Figure 44B ). Neutralizing antibody titers are generally associated with inhibition of viral replication in the lungs and other respiratory sites and are thus associated with protective efficacy in a subject. Thus, without wishing to be bound by theory, the cytoplasmic tail deletions disclosed herein may allow for protective efficacy against RSV to be achieved at lower doses of nucleic acid-based vaccines, resulting in further possible benefits such as reduced reactogenicity.

[0311] In embodiments in which the RSV-F protein comprises a cytoplasmic tail deletion (as defined in this subsection and the appended numbered embodiments and claims), when the F1 domain and transmembrane domain of the RSV-F protein are aligned with positions 137-549 of SEQ ID NO: 107 or 108, an RSV-F protein having a "cytoplasmic tail" refers to the presence of residues (e.g., 5 residues) at the C-terminus of the residue aligned with position 549 (Y) of SEQ ID NO: 107 or 108. Thus, the cytoplasmic tail is at the C-terminus of the transmembrane domain. Preferably, an RSV-F protein having a "cytoplasmic tail" refers to the presence of residues (e.g., 5 residues) at the C-terminus of position 549 of the RSV-F protein. For example, the RSV-F construct designated ΔCT25 used in the examples (see, e.g., Table 8) does not contain any residues at the C-terminus of Y at position 549 and thus does not contain a cytoplasmic tail. Reference to deletion of, e.g., 2-20 residues from the C-terminus of the CT (or the like) (relative to SEQ ID NO: 109 or 110) refers to deletion of at least these two and no more than these 20 most C-terminal residues from the CT. That is, deletion of at least the C-terminal residues SN or SK relative to SEQ ID NO: 109 or 110, respectively, and no more than deletion of the C-terminal residues TPVTLSKDQLSGINNIAFSN or TPVTLSKDQLSGINNIAFSK relative to SEQ ID NO: 109 or 110, respectively.

[0312] In some embodiments, the nucleic acids of the present disclosure encode an RSV-F protein comprising a cytoplasmic tail; wherein, relative to the cytoplasmic tail according to SEQ ID NO: 109 or 110, 2-20 residues are deleted from the C-terminus of the cytoplasmic tail of the RSV-F protein. In some embodiments, 3-20 residues are deleted from the C-terminus. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or at least 19 residues are deleted from the C-terminus. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues are deleted from the C-terminus. In some embodiments, 2-5, 3-5, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, or 15-20 residues are deleted from the C-terminus.

[0313] In a preferred embodiment, 2-5, such as 2-4, 2-3, or 3-4, and preferably 3 residues are deleted from the C-terminus of the CT of the RSV-F protein (relative to the wild-type cytoplasmic tail according to SEQ ID NO: 109 or 110). As illustrated, for example, in Example 12B (see Figure 39), the deletion of these 3 C-terminal residues (“ΔCT3”) enhanced the expression of cell surface trimeric pre-fusion RSV-F from the nucleic acid (as measured by AM14 antibody binding) during the 96-hour period after transfection, relative to the expression of the parental molecule with intact CD or complete CD deletion. This enhanced expression phenotype was observed for all four RSV-F constructs (F318, F319, F(i), and F(ii)) tested. See also, for example, Example 12E( Figure 42A) which uses the "ΔCT5" construct. In a preferred embodiment, the cytoplasmic tail comprises or consists of: (i) the amino acid sequence of positions 10-31 according to SEQ ID NO: 134, or (ii) an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally of the same length as said positions; and wherein the cytoplasmic tail does not contain any residues at the C-terminus of the amino acid sequence of (i) or (ii). In another embodiment, the cytoplasmic tail comprises or consists of: (i) the amino acid sequence of positions 10-29 according to SEQ ID NO: 135, or (ii) an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally of the same length as said positions; and wherein the cytoplasmic tail does not contain any residues at the C-terminus of the amino acid sequence of (i) or (ii).

[0314] In a preferred embodiment, 6-13, such as 7-13, 8-12, 9-11, 9-10 or 10-11, and preferably 10 residues are deleted from the C-terminus of the CT of the RSV-F protein (relative to the wild-type cytoplasmic tail according to SEQ ID NO: 109 or 110). As illustrated, for example, in Example 12E (see Figure 42), the deletion of these 10 C-terminal residues ("ΔCT10") enhanced the expression of cell surface pre-fusion RSV-F from nucleic acid (as measured by AM14 antibody binding) relative to the expression of parental molecules with intact CT or complete CT deletion during a 47-hour period after transfection. In a preferred embodiment, the cytoplasmic tail comprises or consists of: (i) the amino acid sequence of positions 10-24 according to SEQ ID NO: 136, or (ii) an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally of the same length as said positions; and wherein the cytoplasmic tail does not contain any residues at the C-terminus of the amino acid sequence of (i) or (ii).

[0315] In a preferred embodiment, 14 - 16, such as 14 - 15 or 15 - 16, and preferably 15 residues are deleted from the C-terminus of the CT of the RSV-F protein (relative to the wild-type cytoplasmic tail according to SEQ ID NO: 109 or 110). As illustrated, for example, in Example 12E (see Figure 42), during the 47-hour period after transfection, deletion of these 15 C-terminal residues ("ΔCT15") enhanced the expression of cell surface trimeric pre-fusion RSV-F from nucleic acid (as measured by AM14 antibody binding) relative to the expression of parental molecules with an intact CT or a completely deleted CT. In a preferred embodiment, the cytoplasmic tail comprises or consists of: (i) the amino acid sequence of positions 10 - 19 according to SEQ ID NO: 137, or (ii) an amino acid sequence that is at least 60%, 70%, 80%, or 90% identical to said positions and optionally of the same length as said positions; and wherein the cytoplasmic tail does not contain any residues at the C-terminus of the amino acid sequence of (i) or (ii).

[0316] In a more preferred embodiment, 16 - 20, such as 17 - 20, 18 - 20, or 19 - 20, and preferably 20 residues are deleted from the C-terminus of the CT of the RSV-F protein (relative to the wild-type cytoplasmic tail according to SEQ ID NO: 109 or 110). As illustrated, for example, in Example 12B (see Figure 39), during the 96-hour period after transfection, deletion of these 20 C-terminal residues ("ΔCT20") enhanced the expression of cell surface trimeric pre-fusion RSV-F from nucleic acid (as measured by AM14 antibody binding) relative to the expression of parental molecules with an intact CT or a completely deleted CT. This expression was also enhanced compared to the "ΔCT3" construct, and this phenotype was observed for all four RSV-F constructs (F318, F319, F(i), and F(ii)) tested. Also see, for example, Example 12E( Figure 42A ), where the greatest trimeric pre-fusion expression was observed for the ΔCT20 construct. Also see, for example, Example 13, where at a low dose of RNA (0.2 μg), constructs with ΔCT20 tended to elicit a more potent neutralizing antibody response in vivo compared to their parental molecules with a completely intact CT (see, for example, Figure 44B)。Therefore, deletions of 16 - 20, such as 17 - 20, 18 - 20, or 19 - 20 C - terminal residues, and particularly the deletion of these 20 C - terminal residues, are more preferred than deletions of other numbers of residues from the C - terminus. In another more preferred embodiment, the cytoplasmic tail comprises or consists of: (i) the amino acid sequence at positions 10 - 14 of SEQ ID NO:138, or (ii) an amino acid sequence that is at least 60% or 80% identical to said positions and optionally has the same length as said positions; and wherein the cytoplasmic tail does not include any residues at the C - terminus of the amino acid sequence in (i) or (ii).

[0317] Generally, relative to an RSV - F protein having the same amino acid sequence but lacking the deletion (e.g., comprising a wild - type cytoplasmic tail, e.g., according to SEQ ID NO:109 or 110), the deletions outlined above increase the cell - surface expression of the RSV - F protein from RNA (e.g., within at least 24, 48, 72, or 96 hours; or e.g., within 24, 48, 72, or 96 hours). Generally, relative to the expression of an RSV - F protein having the same amino acid sequence but lacking such deletions (e.g., comprising a wild - type cytoplasmic tail, e.g., according to SEQ ID NO:109 or 110) in this form, the deletions outlined above increase the cell - surface expression of the RSV - F protein in a pre - fusion trimeric form from RNA (e.g., within at least 24, 48, 72, or 96 hours; or e.g., within 24, 48, 72, or 96 hours).

[0318] When determining the effect of cytoplasmic tail deletions, AM14 antibody binding (or alternatively defined as, binding of an antibody comprising the light chain (LC) according to SEQ ID NO:2 and the heavy chain (HC) according to SEQ ID NO:3) is typically used to evaluate pre - fusion trimeric RSV - F expression. AM14 antibody binding can be assayed using indirect immunofluorescence labeling, e.g., using the protocol in the Examples (see subsection "Indirect Immunofluorescence Labeling and Detection of Surface - Expressed RSV F"). Cell - surface expression can be evaluated in fibroblasts, preferably human fibroblasts, preferably human foreskin fibroblasts, preferably human primary BJ cells, preferably the CRL - 2522 cell line (deposited with the American Type Culture Collection (ATCC) under said accession number and publicly available).

[0319] General characteristics of nucleic acid

[0320] The nucleic acids of the present disclosure can be DNA or RNA (including their hybrids), preferably RNA. DNA and RNA analogs, such as those containing modified backbones (e.g., peptide nucleic acids (PNA) or phosphorothioates) or modified bases, are within the scope of the present disclosure. The nucleic acid can be linear, circular, and / or branched, but will generally be linear. Typically, the nucleic acid will be in recombinant form, i.e., a form not found in nature.

[0321] The nucleic acid can be used to express the RSV-F protein of the present disclosure in vitro from a host cell (i.e., the nucleic acid is an expression vector or a part thereof). Suitable nucleic acid expression vectors (in particular, DNA expression vectors) can comprise, for example, (1) an origin of replication; (2) a selectable marker gene; (3) one or more expression control elements, such as transcriptional control elements (e.g., promoters, enhancers, or terminators), and / or one or more translation signals; and (4) a signal sequence or leader sequence for targeting to the secretory pathway in a selected host cell (e.g., those sequences detailed above in the section entitled "Preparation of RSV-F Protein"). In a preferred alternative embodiment, the nucleic acid is used to express the RSV-F protein of the present disclosure in vivo in a subject (i.e., the nucleic acid is a nucleic acid-based vaccine or a part thereof). In such preferred embodiments, in addition to the sequence encoding the RSV-F protein of the present disclosure, the nucleic acid can comprise one or more heterologous sequences, such as sequences encoding other proteins (e.g., as detailed below) and / or control sequences, in particular promoters or internal ribosome entry sites.

[0322] The nucleic acids of the present disclosure can be codon-optimized. In some embodiments, the nucleic acids of the present disclosure can be codon-optimized for expression in human cells. Codon optimization refers to the use of specific codons that can increase the translation efficiency and / or half-life of the nucleic acid while not changing the sequence of the expressed protein (taking into account genetic code redundancy). Embodiments of codon-optimized RNA are discussed in more detail in the subsection entitled "RNA" below.

[0323] In some embodiments, the nucleic acids of the present disclosure are in the form of viral vectors, such as replication-competent viral vectors or replication-deficient viral vectors; including viral vectors based on both DNA and RNA. Suitable examples of viral vectors for encoding the RSV-F protein of the present disclosure include, for example: adenoviral vectors, such as replication-deficient or replication-competent adenoviral vectors; poxviral vectors, such as vaccinia virus vectors (e.g., modified vaccinia Ankara virus (MVA), NYVAC, avipox vectors, canarypox (e.g., ALVAC) and fowl pox virus (FPV)); alphaviral vectors, such as Sindbis virus, Semlike Forest virus (SFV), Ross River virus, Venezuelan equine encephalitis (VEE) virus, and chimeras derived from alphaviral vectors, such as the foregoing viruses; herpesviral vectors, such as vectors derived from cytomegalovirus (CMV); arenaviral vectors, such as lymphocytic choriomeningitis virus (LCMV) vectors; measles virus vectors; vesicular stomatitis virus vectors; pseudorabies virus vectors; adeno-associated virus vectors; retroviral vectors; lentiviral vectors; and virus-like particles. In other embodiments, the nucleic acid is in the form of a DNA plasmid.

[0324] In embodiments in which the nucleic acids of the present disclosure are viral vectors, preferably the viral vector is an adenovirus vector, such as a replication-incompetent adenovirus type 26 (“Ad26”) or a replication-incompetent chimpanzee adenovirus 155 (“ChAd155”), preferably replication-incompetent Ad26. In such adenovirus vector embodiments, a particular group of patients of interest (wherein the adenovirus can be used for therapy, particularly vaccination) is infants and the elderly (see the section entitled “Medical Use and Methods of Treatment” below). In such adenovirus vector embodiments, the adenovirus vector (preferably replication-incompetent Ad26) can also be co-formulated with the RSV-F protein of the present disclosure (i.e., the protein itself), which can have the same or a different primary amino acid sequence as the RSV-F protein of the present disclosure encoded by the adenovirus. In such adenovirus vector embodiments, alternatively, the adenovirus vector (preferably replication-incompetent Ad26) can be co-formulated with other RSV-F proteins (i.e., the protein itself, which is not the RSV-F protein according to the present disclosure), such as an RSV-F protein with a p27 region deletion (or without a p27 region deletion), and optionally having at least 2, 3, 4, or 5 mutations relative to wild-type RSV-F (such as N67I and S215P; N67I, S215P, and E487Q; or K66E, N67I, I76V, S215P, and D486N; particularly the latter set of five mutations). In such co-formulation embodiments, a particular group of patients of interest (wherein the co-formulation can be used for therapy, particularly vaccination) is the elderly (see the section entitled “Medical Use and Methods of Treatment” below). In such elderly patients, the co-formulation can be administered as an initial boosting regimen or as part thereof, particularly involving administering the co-formulation as both an (one or more) initial administration and an (one or more) boosting administration.

[0325] The nucleic acid (preferably RNA) can encode only the RSV-F protein of the present disclosure (i.e., the nucleic acid encodes a single protein). Alternatively, the nucleic acid can encode multiple proteins, one of which is the RSV-F protein of the present disclosure. In some embodiments, the nucleic acid encodes at least (i) the RSV-F protein of the present disclosure; and (ii) at least one other protein. The at least one other protein can be a nanoparticle, such as a ferritin nanoparticle (e.g., which, together with the RSV-F protein of the present disclosure, is encoded by a single open reading frame, resulting in the expression of a single polypeptide). In a preferred embodiment, the at least one other protein is an antigen; and thus can comprise or can be a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a tumor antigen, or an allergenic (i.e., derived from or originating from an allergen) antigen; typically encoded by a separate open reading frame of the RSV-F protein of the present invention. The at least one other protein will generally be a pathogen antigen. The at least one other protein will generally be an antigen that is a surface polypeptide, such as a spike glycoprotein, a hemagglutinin, an adhesin, or an envelope glycoprotein. In a specific embodiment, the at least one other protein is an antigen from or derived from a virus (especially a virus that causes respiratory diseases, especially a seasonal virus that causes respiratory diseases). In embodiments in which the at least one other protein is an antigen from or derived from a virus, examples of such viruses include: Coronavirus, Orthomyxovirus, Pneumoviridae, Paramyxoviridae, Poxviridae, Picornavirus, Bunyavirus, Heparnavirus, Filovirus, Togavirus, Flavivirus, Pestivirus, Hepadnavirus, Rhabdovirus, Caliciviridae, Retrovirus, Reovirus, Parvovirus, Herpesvirus, Papovaviruses, and Adenovirus.

[0326] In a preferred embodiment, the at least one other protein detailed above is a paramyxovirus protein (especially a paramyxovirus antigen). Useful paramyxovirus proteins (especially, antigens) can be from orthoparamyxovirus or metaparamyxovirus, especially human RSV or human metaparamyxovirus (hMPV). Useful other hMPV antigens include, for example, F, N, P, M, M2-1 and M2 antigens (especially, F antigen). Such hMPV proteins (especially, antigens) can be from or derived from subtype A or B. In a preferred embodiment, the nucleic acid is an RNA encoding the RSV-F protein of the present disclosure in addition to an hMPV antigen (especially, F antigen). In such RNA embodiments, the preferred patient group (wherein the RNA can be used for therapy, especially vaccination) is infants (see the section entitled "Medical Use and Treatment Methods" below). In addition to other RSV-F antigens (i.e., having a different amino acid sequence from the RSV-F protein of the present disclosure encoded by the nucleic acid), useful other human RSV antigens include, for example, G, M1, M2-1, M2-2, P, L, N, NS1, NS2 and SH antigens. Such other human RSV proteins (especially, antigens; especially, F antigen) can be from or derived from subtype A or B. In a specific embodiment, the nucleic acid is a viral vector (especially, a poxvirus vector, especially an MVA vector) encoding the RSV-F protein of the present disclosure in addition to a plurality of other RSV proteins (especially, antigens; especially at least 2, 3 or 4 other RSV proteins / antigens; especially selected from G (from or derived from subtype A: "G A "), G (from or derived from subtype B: "G B "), N and either M2-1 or M2-2; especially G A ), G B ), N and either M2-1 or M2-1). In such viral vector embodiments, the specific patient group (wherein the viral vector can be used for therapy, especially vaccination) is the elderly (see the section entitled "Medical Use and Treatment Methods" below).

[0327] In a preferred embodiment, the at least one other protein detailed above is a coronavirus antigen. Useful coronavirus antigens can be from the SARS coronavirus, particularly SARS-CoV2. Useful coronavirus antigens (preferably SARS-CoV2 antigens) include spike, M, E, HE, nucleocapsid, Plpro, and 3CLPro proteins, particularly the spike protein. Preferably, the coronavirus antigen is the SARS-CoV2 spike protein. The SARS-CoV2 spike protein can be from any variant, such as Omicron (e.g., Omicron BA.1, BA.2, BA.3, BA.4, or BA.5), Alpha, Epsilon, Eta, Theta, Kappa, Iota, Zeta, Mu, Lambda, Beta, Gamma, or Delta. Preferably, the SARS-CoV2 spike protein contains one or more mutations relative to the wild-type protein, particularly mutations of one or more (e.g., two) proline residues. The one or more mutations can be introduced to stabilize the SARS-CoV2 spike protein in the pre-fusion conformation. In a preferred embodiment, the nucleic acid is an RNA that encodes the RSV-F protein of the present disclosure in addition to the coronavirus antigen, as detailed above, for example. In such RNA embodiments, the preferred patient group (wherein the RNA can be used for therapy, particularly vaccination) is the elderly (see the section entitled "Medical Use and Methods of Treatment" below).

[0328] In another preferred embodiment, the at least one other protein detailed above is an orthomyxovirus antigen. Useful orthomyxovirus antigens can be from influenza A, B, or C viruses. Useful orthomyxovirus antigens (particularly influenza A, B, or C virus antigens) include hemagglutinin, neuraminidase, and matrix M2 proteins, particularly hemagglutinin. Preferably, the orthomyxovirus antigen is the hemagglutinin of influenza A virus. The hemagglutinin of influenza A virus can be from any subtype, such as H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16. In a preferred embodiment, the nucleic acid is an RNA that encodes the RSV-F protein of the present disclosure in addition to the orthomyxovirus antigen, as detailed above, for example. In such RNA embodiments, the preferred patient group (wherein the RNA can be used for therapy, particularly vaccination) is the elderly (see the section entitled "Medical Use and Methods of Treatment" below). In such RNA embodiments, the RNA can encode (i) the RSV-F protein of the present disclosure, (ii) a coronavirus antigen, as detailed above, for example, and (iii) an orthomyxovirus antigen, as detailed above.

[0329] In particular, the various nucleic acids of the present disclosure are provided in purified or substantially purified form; that is, substantially free of other nucleic acids (e.g., free or substantially free of naturally occurring nucleic acids, such as other nucleic acids expressed by a host cell). The various nucleic acids are generally at least 50% pure (by weight), such as at least 60%, 70%, 80%, 90% or 95% pure (by weight).

[0330] In a further independent aspect, the present disclosure also provides a vector comprising one or more nucleic acids of the present disclosure.

[0331] The nucleic acid encoding the RSV-F protein of the present disclosure can be delivered naked or preferably in combination with a vector (e.g., as detailed in the section entitled "Vectors Comprising Nucleic Acids Encoding RSV-F Proteins in the Pre-Fusion Conformation" below).

[0332] Generally, the nucleic acids of the present disclosure (preferably RNA) and the RSV-F protein encoded thereby elicit a pre-fusion RSV-F specific antibody response against RSV in vivo, such as an IgG antibody response (see, e.g., Examples 11 and 13).

[0333] Generally, the nucleic acids of the present disclosure (preferably RNA) and the RSV-F protein encoded thereby elicit a neutralizing antibody response against RSV in vivo, such as a neutralizing antibody response against RSV-A (see, e.g., Examples 11 and 13). The neutralizing antibody response can inhibit the replication of RSV in the respiratory system of a subject (such as in the lungs). The neutralizing antibody response can generate protective immunity against RSV in a subject.

[0334] RNA

[0335] In a preferred embodiment, the nucleic acid of the present disclosure (encoding the RSV-F protein of the present disclosure) is RNA.

[0336] In the context of the section entitled "RNA" herein, "RNA" refers to an artificial (or alternatively defined as recombinant) ribonucleic acid encoding the RSV-F protein of the present disclosure, which can be translated in a cell (i.e., mRNA). Preferably, the RNA is neither a viral vector or a virus-based vaccine (such as a live attenuated virus vaccine), nor does it contain one within it.

[0337] RNA molecules can have different lengths, but are typically 500 - 20,000 ribonucleotides long, such as 1000 - 20,000, 1000 - 15,000, 1000 - 10,000, 1000 - 5000, 1000 - 3000, 1000 - 2500, 1000 - 2500 or 1000 - 2000 ribonucleotides long. The RNA can be non-self-replicating (also referred to as "conventional" RNA), or self-replicating; preferably non-self-replicating.

[0338] In some embodiments, the RNA is self-replicating. Self-replicating RNA can be produced using replication elements derived from, for example, alphaviruses and replacing the sequence encoding the structural viral protein with a sequence encoding at least one RSV-F protein of the present disclosure. Self-replicating RNA molecules are typically positive-strand molecules, which can be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase, which then produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA results in the production of multiple daughter RNAs. These daughter RNAs, as well as the collinear subgenomic transcripts themselves, can be translated to provide in situ expression of the encoded protein (i.e., the RSV-F protein of the present disclosure); or can be transcribed to provide other transcripts having the same sense strand as the delivered RNA, which are translated to provide in situ expression of the encoded protein. The overall result of this transcriptional sequence is a significant amplification of the amount of RNA introduced, and thus the encoded RSV-F protein of the present disclosure (potentially in addition to other proteins as detailed above) becomes the major polypeptide product of the cell.

[0339] In such embodiments in which the RNA self-replicates, the RNA can encode (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-replicating RNA, and (ii) the RSV-F protein of the present disclosure. The polymerase can be an alphavirus replicase, such as comprising one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP4. Such alphavirus-based self-replicating RNAs can use replicases from, for example, Sindbis virus, Semliki Forest virus, eastern equine encephalitis virus (EEEV), or Venezuelan equine encephalitis virus (VEEV). Mutant or wild-type virus sequences can be used; for example, the attenuated TC83 mutant of VEEV has been used for self-replicating RNAs (see

[22] ). Thus, the self-replicating RNA encoding the RSV-F protein of the present disclosure can have two open reading frames. The first (5') open reading frame encodes the replicase, particularly an alphavirus replicase (e.g., as detailed above); the second (3') open reading frame encodes the RSV-F protein of the present disclosure. There can also be other open reading frames encoding (i) one or more other proteins (preferably one or more other antigens, e.g., as detailed above); and / or (ii) accessory polypeptides.

[0340] Generally, the RNA comprises a 5' cap, such as 7'-methylguanosine (also known as 7-methylguanosine / m 7 G / m7G), which can be added by enzymatic means or non-enzymatic reaction. The RNA can have the following exemplary 5' caps:

[0341] - 7'-methylguanosine linked 5' to 5' to the 5' first ribonucleotide via a triphosphate bridge (also known as "CapO");

[0342] - 7'-methylguanosine linked 5' to 5' to the 5' first ribonucleotide via a triphosphate bridge, and wherein the first 5' ribonucleotide comprises 2'-methylated ribose (2'-O-Me) (also known as "Cap 1");

[0343] - 7'-methylguanosine linked 5' to 5' to the 5' first ribonucleotide via a triphosphate bridge, and wherein the first 5' ribonucleotide and the second 5' ribonucleotide comprise 2'-methylated ribose (2'-O-Me) (also known as "Cap 2");

[0344] - or 7'-methylguanosine linked 5' to 5' to the 5' first ribonucleotide via a triphosphate bridge, and wherein the first, second, and third 5' ribonucleotides comprise 2'-methylated ribose (2'-O-Me).

[0345] In a preferred embodiment, the 5' cap is a 7'-methylguanosine linked 5' to 5' via a triphosphate bridge to the 5' first ribonucleoside, and wherein the first 5' ribonucleoside comprises a 2'-methylated ribose (2'-O-Me), e.g., the 5' end of the RNA has the structure m7G(5')ppp(5')(2'OMeA)pG. Preferably, such a cap is added non-enzymatically using the following reagent:

[0346]

[0347] The reagent is sold as CLEANCAP Reagent AG (TRILINK BIOTECHNOLOGIES).

[0348] In other embodiments, a cap can be added such that the 5' end of the RNA has the structure m7(3'OMeG)(5')ppp(5')(2'OMeA)pG. Such a cap can be added non-enzymatically using the following reagent:

[0349]

[0350] The reagent is sold as CLEANCAP Reagent AG (3'OMe) (TRILINK BIOTECHNOLOGIES).

[0351] Generally, the RNA comprises a 3' polyadenosine ("polyA") tail, e.g., comprising 10 - 700 A ribonucleotides. The polyA tail may comprise at least two stretches of contiguous A ribonucleotides (also referred to as "split polyA tail") that are non - contiguous, or (in particular, only one) a stretch of contiguous A ribonucleotides. The total number of A ribonucleotides ("A") in at least two non - contiguous stretches may be, e.g., 10 - 700, such as 10 - 600, 10 - 500, 20 - 500, 50 - 500, 70 - 500, 100 - 500, 20 - 400, 30 - 300, 40 - 200, 50 - 150, 70 - 120, 100 - 120, or in particular 100 - 120. The total number of A in (in particular, only one) contiguous stretch may be, e.g., 10 - 700; such as 10 - 600, 20 - 600, or in particular 40 - 600 (e.g., 50 - 600, 80 - 600, 80 - 550, 100 - 500; or 40 - 70, 50 - 65 or 55 - 65). Where at least two non - contiguous stretches of A are used, these stretches may have different lengths. For example, the length of the first stretch may be 10 - 150 A, such as 10 - 100, 10 - 50, 15 - 50, 20 - 50, 20 - 40, 25 - 40, or in particular 25 - 35 A. For example, the length of the second stretch may be 10 - 150 A, such as 10 - 150, 20 - 120, 30 - 100, 40 - 90, 50 - 90, 60 - 90, 65 - 90, 70 - 90, or in particular 80 - 90 A. The first stretch may be located 5' or 3' relative to the second stretch. However, in a specific embodiment, the first stretch is located 5' relative to the second stretch. In a further specific embodiment, the polyA tail comprises, in the 5' to 3' direction, a first non - contiguous stretch of A and a second non - contiguous stretch of A, having lengths of 25 - 35 and 80 - 90 A, respectively. In a further specific embodiment, the polyA tail comprises, in the 5' to 3' direction, a first non - contiguous stretch of A and a second non - contiguous stretch of A, having lengths of 25 - 35 and 65 - 90 A, respectively. In some embodiments, the at least two non - contiguous stretches of A are from or are part of the 3' untranslated region (UTR), e.g., as detailed below.

[0352] The RNA preferably comprises (in addition to any 5' cap structure) one or more modified ribonucleotides, i.e., ribonucleotides that are structurally modified relative to the standard A, C, G, or U ribonucleotides. In other embodiments, the RNA does not comprise modified ribonucleotides, i.e., the RNA contains only standard A, C, G, or U ribonucleotides (except for any 5' cap structure (if present), e.g., as detailed above). In preferred embodiments in which one or more modified ribonucleotides are used, the one or more modified ribonucleotides can be or can comprise N1-methylpseudouridine ("1mΨ"); pseudouridine ("Ψ"); N1-ethylpseudouridine; 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine (m 6A); N6-threonylcarbamoyladenosine; 1,2'-O-dimethyladenosine; 1-methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; 2-methylthio-N6-hydroxy-norvalylcarbamoyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine; N6,2'-O-dimethyladenosine; N6,2'-O-dimethyladenosine; N6,N6,2'-O-trimethyladenosine; N6,N6-dimethyladenosine; N6-acetyladenosine; N6-hydroxy-norvalylcarbamoyladenosine; N6-methyl-N6-threonylcarbamoyladenosine; 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; 7-deazaadenosine; N1-methyladenosine; N6,N6(dimethyl)adenine; N6-cis-hydroxyisopentenyladenosine; α-thioadenosine; 2(amino)adenine; 2(aminopropyl)adenine; 2(methylthio)N6(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine; 2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine; 2-(propyl)adenine; 2'-amino-2'-deoxy-ATP; 2'-azido-2'-deoxy-ATP; 2'-deoxy-2'-α-aminoadenosine TP; 2'-deoxy-2'-α-azidoadenosine TP; 6(alkyl)adenine; 6(methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine; 7(deaza)adenine; 8(alkenyl)adenine; 8(alkynyl)adenine; 8(amino)adenine; 8(thioalkyl)adenine; 8-(alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine; 8-(hydroxy)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azidoadenosine; azidoadenine; deazaadenine; N6(methyl)adenine; N6-(isopentyl)adenine; 7-deaza-8-azidoadenosine; 7-methyladenine; 1-deazaadenosine TP; 2'-fluoro-N6-Bz-deoxyadenosine TP; 2'-OMe-2-amino-ATP; 2'O-methyl-N6-Bz-deoxyadenosine TP; 2'-α-ethynyladenosine TP; 2-aminoadenine; 2-aminoadenosine TP; 2-amino-ATP; 2'-α-trifluoromethyladenosine TP; 2-azidoadenosine TP; 2'-β-ethynyladenosine TP; 2-bromoadenosine TP; 2'-β-trifluoromethyladenosine TP; 2-chloroadenosine TP; 2'-deoxy-2',2'-difluoroadenosine TP; 2'-deoxy-2'-α-mercaptoadenosine TP; 2'-deoxy-2'-α-thiomethoxyadenosine TP; 2'-deoxy-2'-β-aminoadenosine TP; 2'-deoxy-2'-β-azidoadenosine TP;2'-Deoxy-2'-β-bromoadenosine TP; 2'-Deoxy-2'-β-chloroadenosine TP; 2'-Deoxy-2'-β-fluoroadenosine TP; 2'-Deoxy-2'-β-iodoadenosine TP; 2'-Deoxy-2'-β-mercaptoadenosine TP; 2'-Deoxy-2'-β-thiomethoxyadenosine TP; 2-Fluoroadenosine TP; 2-Iodoadenosine TP; 2-Mercaptoadenosine TP; 2-Methoxyadenine; 2-Methylthioadenine; 2-Trifluoromethyladenosine TP; 3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP; 3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP; 3-Deazaadenosine TP; 4'-Azidoadenosine TP; 4'-Carbocyclic adenosine TP; 4'-Ethynyladenosine TP; 5'-Homo-adenosine TP; 8-Aza-ATP; 8-Bromoadenosine TP; 8-Trifluoromethyladenosine TP; 9-Deazaadenosine TP; 2-Aminopurine; 7-Deaza-2,6-diaminopurine; 7-Deaza-8-aza-2,6-diaminopurine; 7-Deaza-8-aza-2-aminopurine; 2,6-Diaminopurine; 7-Deaza-8-azaadenine, 7-Deaza-2-aminopurine; 2-Thiocytidine; 3-Methylcytidine; 5-Formylcytidine; 5-Hydroxymethylcytidine; 5-Methylcytidine; N4-Acetylcytidine; 2'-O-Methylcytidine; 2'-O-Methylcytidine; 5,2'-O-Dimethylcytidine; 5-Formyl-2'-O-methylcytidine; Lysidine; N4,2'-O-Dimethylcytidine; N4-Acetyl-2'-O-methylcytidine; N4-Methylcytidine; N4,N4-Dimethyl-2'-OMe-cytidine TP; 4-Methylcytidine; 5-Azacytidine; Pseudoisocytidine; Pyrrolo-cytidine; α-Thiocytidine; 2-(Thio)cytosine; 2'-Amino-2'-deoxy-CTP; 2'-Azido-2'-deoxyCTP; 2'-Deoxy-2'-α-aminocytidine TP; 2'-Deoxy-2'-α-azidocytidine TP; 3(Deaza)5(Azido)cytosine; 3(Methyl)cytosine; 3-(Alkyl)cytosine; 3-(Deaza)5(Azido)cytosine; 3-(Methyl)cytidine; 4,2'-O-Dimethylcytidine; 5(Halo)cytosine; 5(Methyl)cytosine; 5(Propargyl)cytosine; 5(Trifluoromethyl)cytosine; 5-(Alkyl)cytosine; 5-(Alkynyl)cytosine; 5-(Halo)cytosine; 5-(Propargyl)cytosine; 5-(Trifluoromethyl)cytosine; 5-Bromocytidine; 5-Iodocytidine; 5-Propargylcytosine; 6-(Azido)cytosine; 6-Azacytidine; Azidocytosine; Deazacytosine; N4(Acetyl)cytosine; 1-Methyl-1-deazapseudoisocytidine; 1-Methylpseudoisocytidine; 2-Methoxy-5-methylcytidine; 2-Methoxycytidine; 2-Thio-5-methylcytidine; 4-Methoxy-1-methylpseudoisocytidine; 4-Methoxypseudoisocytidine; 4-Thio-1-methyl-1-azapseudoisocytidine; 4-Thio-1-methylpseudoisocytidine;4-Thio-pseudoisocytidine; 5-Aza-zebularine; 5-Methyl-zebularine; Pyrrolo-pseudoisocytidine; Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2'-Anhydrocytidine TP Hydrochloride; 2'-Fluoro-N4-Bz-cytidine TP; 2'-Fluoro-N4-Acetylcytidine TP; 2'-O-Methyl-N4-Acetylcytidine TP; 2'O-Methyl-N4-Bz-cytidine TP; 2'-α-Ethynylcytidine TP; 2'-α-Trifluoromethylcytidine TP; 2'-β-Ethynylcytidine TP; 2'-β-Trifluoromethylcytidine TP; 2'-Deoxy-2',2'-Difluorocytidine TP; 2'-Deoxy-2'-α-Mercaptocytidine TP; 2'-Deoxy-2'-α-Thiomethoxy-cytidine TP; 2'-Deoxy-2'-β-Aminocytidine TP; 2'-Deoxy-2'-β-Azacytidine TP; 2'-Deoxy-2'-β-Bromocytidine TP; 2'-Deoxy-2'-β-Chlorocytidine TP; 2'-Deoxy-2'-β-Fluorocytidine TP; 2'-Deoxy-2'-β-Iodocytidine TP; 2'-Deoxy-2'-β-Mercaptocytidine TP; 2'-Deoxy-2'-β-Thiomethoxy-cytidine TP; 2'-O-Methyl-5-(1-Propynyl)cytidine TP; 3'-Ethynylcytidine TP; 4'-Azacytidine TP; 4'-Carbocyclic cytidine TP; 4'-Ethynylcytidine TP; 5-(1-Propynyl)Ara-Cytidine TP; 5-(2-Chlorophenyl)-2-Thiocytidine TP; 5-(4-Aminophenyl)-2-Thiocytidine TP; 5-Aminoallyl-CTP; 5-Cyanocytidine TP; 5-Ethynyl-Ara-Cytidine TP; 5-Ethynylcytidine TP; 5'-Homo-cytidine TP; 5-Methoxycytidine TP; 5-Trifluoromethylcytidine TP; N4-Aminocytidine TP; N4-Benzoyl-cytidine TP; Pseudoisocytidine; 7-Methylguanosine; N2,2'-O-Dimethylguanosine; N2-Methylguanosine; Wyosine; 1,2'-O-Dimethylguanosine; 1-Methylguanosine; 2'-O-Methylguanosine; 2'-O-Ribosylguanosine (Phosphate); 2'-O-Methylguanosine; 2'-O-Ribosylguanosine (Phosphate); 7-Aminomethyl-7-Deazaguanosine; 7-Cyano-7-Deazaguanosine; Archaeosine; Methylwyosine; N2,7-Dimethylguanosine; N2,N2,2'-O-Trimethylguanosine; N2,N2,7-Trimethylguanosine; N2,N2-Dimethylguanosine; N2,7,2'-O-Trimethylguanosine; 6-Thioguanosine; 7-Deazaguanosine; 8-Oxoguanosine; N1-Methylguanosine; α-Thioguanosine; 2(Propyl)Guanine; 2-(Alkyl)Guanine; 2'-Amino-2'-Deoxy-GTP; 2'-Azido-2'-Deoxy-GTP; 2'-Deoxy-2'-α-Aminoguanosine TP; 2'-Deoxy-2'-α-Azaguanosine TP; 6(Methyl)Guanine; 6-(Alkyl)Guanine; 6-(Methyl)Guanine;6-Methylguanosine; 7(alkyl)guanine; 7(denitrogenated)guanine; 7(methyl)guanine; 7-(alkyl)guanine; 7-(denitrogenated)guanine; 7-(methyl)guanine; 8(alkyl)guanine; 8(alkynyl)guanine; 8(halogenated)guanine; 8(thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halogenated)guanine; 8-(hydroxy)guanine; 8-(thioalkyl)guanine; 8-(mercapto)guanine; Azaguanine; Denitrogenated guanine; N(methyl)guanine; N-(methyl)guanine; 1-Methyl-6-thioguanosine; 6-Methoxyguanosine; 6-Thio-7-denitrogenated-8-azaguanosine; 6-Thio-7-denitrogenated guanosine; 6-Thio-7-methylguanosine; 7-Denitrogenated-8-azaguanosine; 7-Methyl-8-oxoguanosine; N2,N2-Dimethyl-6-thioguanosine; N2-Methyl-6-thioguanosine; 1-Me-GTP; 2’ Fluoro-N2-isobutylguanosine TP; 2’O-Methyl-N2-isobutylguanosine TP; 2’-a-Ethynylguanosine TP; 2’-a-Trifluoromethylguanosine TP; 2’-b-Ethynylguanosine TP; 2’-b-Trifluoromethylguanosine TP; 2’-Deoxy-2’,2’-difluoroguanosine TP; 2’-Deoxy-2’-a-mercapto guanosine TP; 2’-Deoxy-2’-a-thiomethoxy guanosine TP; 2’-Deoxy-2’-b-amino guanosine TP; 2’-Deoxy-2’-b-azido guanosine TP; 2’-Deoxy-2’-b-bromo guanosine TP; 2’-Deoxy-2’-b-chloro guanosine TP; 2’-Deoxy-2’-b-fluoro guanosine TP; 2’-Deoxy-2’-b-iodo guanosine TP; 2’-Deoxy-2’-b-mercapto guanosine TP; 2’-Deoxy-2’-b-thiomethoxy guanosine TP; 4’-Azidoguanosine TP; 4’-Carbocyclic guanosine TP; 4’-Ethynylguanosine TP; 5’-Homo-guanosine TP; 8-Bromoguanosine TP; 9-Denitrogenated guanosine TP; N2-Isobutylguanosine TP; 1-Methylinosine; Inosine; 1,2’-O-Dimethylinosine; 2’-O-Methylinosine; 7-Methylinosine; 2’-O-Methylinosine; Epoxyqueuosine; Galactosyl queuosine; Mannosyl queuosine; Queuosine; Allylaminothymidine; Azidothymidine; Denitrogenated thymidine; Deoxythymidine; 2’-O-Methyluridine; 2-Thiouridine; 3-Methyluridine; 5-Carboxymethyluridine; 5-Hydroxyuridine; 5-Methyluridine; 5-Taurinomethyl-2-thiouridine; 5-Taurinomethyluridine; Dihydrouridine; (3-(3-Amino-3-carboxypropyl)uridine; 1-Methyl-3-(3-amino-5-carboxypropyl)pseudouridine; 1-Methylpseudouridine; 1-Methylpseudouridine; 2’-O-Methyluridine; 2’-O-Methylpseudouridine; 2’-O-Methyluridine; 2-Thio-2’-O-methyluridine;3-(3-Amino-3-carboxypropyl)uridine; 3,2’-O-dimethyluridine; 3-methylpseudouridine TP; 4-thiouridine; 5-(carboxymethyl)uridine; 5-(carboxymethyl)uridine methyl ester, 5,2’-O-dimethyluridine; 5,6-dihydrouridine; 5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2’-O-methyluridine; 5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine; 5-carboxyhydroxymethyluridine methyl ester, 5-carboxymethylaminomethyl-2’-O-methyluridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine; 5-carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2’-O-methyluridine; 5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine; 5-methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-methyldihydrouridine; 5-oxoacetic acid uridine TP; 5-oxoacetic acid methyl ester uridine TP; N1-methylpseudouridine; N1-ethylpseudouridine; uridine 5-oxoacetic acid; uridine 5-oxoacetic acid methyl ester; 3-(3-Amino-3-carboxypropyl)-uridine TP; 5-(isopentenylaminomethyl)-2-thiouridine TP; 5-(isopentenylaminomethyl)-2’-O-methyluridine TP; 5-(isopentenylaminomethyl)uridine TP; 5-propynyluracil; α-thiouridine; 1(aminoalkylaminocarbonylvinyl)-2(thio)-pseudouridine; 1(aminoalkylaminocarbonylvinyl)-2,4-(dithio)pseudouridine; 1(aminoalkylaminocarbonylvinyl)-4(thio)pseudouridine; 1(aminoalkylaminocarbonylvinyl)-pseudouridine; 1(aminocarbonylvinyl)-2(thio)-pseudouridine; 1(aminocarbonylvinyl)-2,4-(dithio)pseudouridine; 1(aminocarbonylvinyl)-4(thio)pseudouridine; 1(aminocarbonylvinyl)-pseudouridine; 1-substituted 2(thio)-pseudouridine; 1-substituted 2,4-(dithio)pseudouridine; 1-substituted 4(thio)pseudouridine; 1-substituted pseudouridine; 1-(aminoalkylaminocarbonylvinyl)-2-(thio)-pseudouridine; 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine TP; 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine UTP; 1-methylpseudouridine UTP; 2(thio)pseudouridine; 2’-deoxyuridine; 2’-fluorouridine; 2-(thio)uracil; 2,4-(dithio)pseudouracil; 2’-methyl, 2’-amino, 2’-azido, 2’-fluoro-guanosine; 2’-amino-2’-deoxy-UTP; 2’-azido-2’-deoxy-UTP; 2’-azido-deoxyuridine TP; 2’-O-methylpseudouridine; 2’-deoxyuridine; 2’-fluorouridine; 2’-deoxy-2’-a-aminouridine TP;2'-deoxy-2'-α-azidouridine TP; 2-methylpseudouridine; 3(3-amino-3-carboxypropyl)uracil; 4(thio)pseudouridine; 4-(thio)pseudouridine; 4-(thio)uracil; 4-thiouridine; 5(1,3-diazol-1-yl)uracil; 5(2-aminopropyl)uracil; 5(aminoalkyl)uracil; 5(dimethylaminoalkyl)uracil; 5(guanidylalkyl)uracil; 5(methoxycarbonylmethyl)-2-(thio)uracil; 5(methoxycarbonyl-methyl)uracil; 5(methyl)2(thio)uracil; 5(methyl)2,4(dithio)uracil; 5(methyl)4(thio)uracil; 5(methylaminomethyl)-2(thio)uracil; 5(methylaminomethyl)-2,4(dithio)uracil; 5(methylaminomethyl)-4(thio)uracil; 5(propynyl)uracil; 5(trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouridine; 5-(alkyl)-2,4(dithio)pseudouridine; 5-(alkyl)-4(thio)pseudouridine; 5-(alkyl)pseudouridine; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil; 5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5-(guanidylalkyl)uracil; 5-(halo)uracil; 5-(1,3-diazol-1-yl)uracil; 5-(methoxy)uracil; 5-(methoxycarbonylmethyl)-2-(thio)uracil; 5-(methoxycarbonyl-methyl)uracil; 5-(methyl)2(thio)uracil; 5-(methyl)2,4(dithio)uracil; 5-(methyl)4(thio)uracil; 5-(methyl)-2-(thio)pseudouridine; 5-(methyl)-2,4(dithio)pseudouridine; 5-(methyl)-4(thio)pseudouridine; 5-(methyl)pseudouridine; 5-(methylaminomethyl)-2(thio)uracil; 5-(methylaminomethyl)-2,4(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5-aminoallyluridine; 5-bromouridine; 5-iodouridine; 5-uracil; 6(aza)uracil; 6-(aza)uracil; 6-azauridine; allylaminouridine; azauracil; deazauracil; N3(methyl)uracil; pseudouridine triphosphate-1-2-acetic acid; pseudouridine; 4-thiopseudouridine triphosphate; 1-carboxymethylpseudouridine; 1-methyl-1-deazapseudouridine; 1-propynyluridine; 1-taurylmethyl-1-methyluridine; 1-taurylmethyl-4-thiouridine; 1-taurylmethylpseudouridine; 2-methoxy-4-thiopseudouridine; 2-thio-1-methyl-1-deazapseudouridine; 2-thio-1-methylpseudouridine; 2-thio-5-azauridine; 2-thiodihydropseudouridine; 2-thiodihydrouridine; 2-thiopseudouridine4-Methoxy-2-thiocytidine; 4-Methoxyuridine; 4-Thio-1-methyluridine; 4-Thiouridine; 5-Azacytidine; Dihydrouridine; (.+-.)1-(2-Hydroxypropyl)uridine TP; (2R)-1-(2-Hydroxypropyl)uridine TP; (2S)-1-(2-Hydroxypropyl)uridine TP; (E)-5-(2-Bromovinyl)arabinouridine TP; (E)-5-(2-Bromovinyl)uridine TP; (Z)-5-(2-Bromovinyl)arabinouridine TP; (Z)-5-(2-Bromovinyl)uridine TP; 1-(2,2,2-Trifluoroethyl)-pseudo-UTP; 1-(2,2,3,3,3-Pentafluoropropyl)uridine TP; 1-(2,2-Diethoxyethyl)uridine TP; 1-(2,4,6-Trimethylbenzyl)uridine TP; 1-(2,4,6-Trimethylbenzyl)pseudo-UTP; 1-(2,4,6-Trimethylphenyl)pseudo-UTP; 1-(2-Amino-2-carboxyethyl)pseudo-UTP; 1-(2-Aminoethyl)pseudo-UTP; 1-(2-Hydroxyethyl)uridine TP; 1-(2-Methoxyethyl)uridine TP; 1-(3,4-Bis(trifluoromethoxy)benzyl)uridine TP; 1-(3,4-Dimethoxybenzyl)uridine TP; 1-(3-Amino-3-carboxypropyl)pseudo-UTP; 1-(3-Aminopropyl)pseudo-UTP; 1-(3-Cyclopropylprop-2-ynyl)uridine TP; 1-(4-Amino-4-carboxybutyl)pseudo-UTP; 1-(4-Aminobenzyl)pseudo-UTP; 1-(4-Aminobutyl)pseudo-UTP; 1-(4-Aminophenyl)pseudo-UTP; 1-(4-Azidobenzyl)uridine TP; 1-(4-Bromobenzyl)uridine TP; 1-(4-Chlorobenzyl)uridine TP; 1-(4-Fluorobenzyl)uridine TP; 1-(4-Iodobenzyl)uridine TP; 1-(4-Methanesulfonylbenzyl)uridine TP; 1-(4-Methoxybenzyl)uridine TP; 1-(4-Methoxybenzyl)pseudo-UTP; 1-(4-Methoxyphenyl)pseudo-UTP; 1-(4-Methylbenzyl)uridine TP; 1-(4-Methylbenzyl)pseudo-UTP; 1-(4-Nitrobenzyl)uridine TP; 1-(4-Nitrobenzyl)pseudo-UTP; 1-(4-Nitrophenyl)pseudo-UTP; 1-(4-Thiomethoxybenzyl)uridine TP; 1-(4-Trifluoromethoxybenzyl)uridine TP; 1-(4-Trifluoromethylbenzyl)uridine TP; 1-(5-Aminopentyl)pseudo-UTP; 1-(6-Aminohexyl)pseudo-UTP; 1,6-Dimethyl-pseudo-UTP; 1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]uridine TP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl}uridine TP; 1-Acetyluridine TP; I-Alkyl-6-(1-propynyl)-pseudo-UTP; 1-Alkyl-6-(2-propynyl)-pseudo-UTP;1-alkyl-6-allyl-psuedo-UTP; 1-alkyl-6-ethynyl-psuedo-UTP; 1-alkyl-6-homoallyl-psuedo-UTP; 1-alkyl-6-vinyl-psuedo-UTP; 1-allyl-psuedouridine TP; 1-aminomethyl-psuedo-UTP; 1-benzoyl-psuedouridine TP; 1-benzyloxymethyl-psuedouridine TP; 1-benzyl-psuedo-UTP; 1-biotinyl-PEG2-psuedouridine TP; 1-biotinyl-psuedouridine TP; 1-butyl-psuedo-UTP; 1-cyanomethyl-psuedouridine TP; 1-cyclobutylmethyl-psuedo-UTP; 1-cyclobutyl-psuedo-UTP; 1-cycloheptylmethyl-psuedo-UTP; 1-cycloheptyl-psuedo-UTP; 1-cyclohexylmethyl-psuedo-UTP; 1-cyclohexyl-psuedo-UTP; 1-cyclooctylmethyl-psuedo-UTP; 1-cyclooctyl-psuedo-UTP; 1-cyclopentylmethyl-psuedo-UTP; 1-cyclopentyl-psuedo-UTP; 1-cyclopropylmethyl-psuedo-UTP; 1-cyclopropyl-psuedo-UTP; 1-ethyl-psuedo-UTP; 1-hexyl-psuedo-UTP; 1-homoallyl-psuedouridine TP; 1-hydroxymethyl-psuedouridine TP; 1-isopropyl-psuedo-UTP; 1-Me-2-thio-psuedo-UTP; 1-Me-4-thio-psuedo-UTP; 1-Me-α-thio-psuedo-UTP; 1-methanesulfonylmethyl-psuedouridine TP; 1-methoxymethyl-psuedouridine TP; 1-methyl-6-(2,2,2-trifluoroethyl)-psuedo-UTP; 1-methyl-6-(4-morpholinyl)-psuedo-UTP; 1-methyl-6-(4-thiomorpholinyl)-psuedo-UTP; 1-methyl-6-(substituted phenyl)-psuedo-UTP; 1-methyl-6-amino-psuedo-UTP; 1-methyl-6-azido-psuedo-UTP; 1-methyl-6-bromo-psuedo-UTP; 1-methyl-6-butyl-psuedo-UTP; 1-methyl-6-chloro-psuedo-UTP; 1-methyl-6-cyano-psuedo-UTP; 1-methyl-6-dimethylamino-psuedo-UTP; 1-methyl-6-ethoxy-psuedo-UTP; 1-methyl-6-ethylcarboxylate-psuedo-UTP; 1-methyl-6-ethyl-psuedo-UTP; 1-methyl-6-fluoro-psuedo-UTP; 1-methyl-6-formyl-psuedo-UTP; 1-methyl-6-hydroxyamino-psuedo-UTP; 1-methyl-6-hydroxy-psuedo-UTP; 1-methyl-6-iodo-psuedo-UTP; 1-methyl-6-isopropyl-psuedo-UTP; 1-methyl-6-methoxy-psuedo-UTP; 1-methyl-6-methylamino-psuedo-UTP; 1-methyl-6-phenyl-psuedo-UTP; 1-methyl-6-propyl-psuedo-UTP; 1-methyl-6-tert-butyl-psuedo-UTP; 1-methyl-6-trifluoromethoxy-psuedo-UTP; 1-methyl-6-trifluoromethyl-psuedo-UTP; 1-morpholinylmethyl-psuedouridine TP; 1-pentyl-pseudo-UTP; 1-phenyl-psuedo-UTP; 1-pivaloyl-psuedouridine TP; 1-propargyl-psuedouridine TP; 1-propyl-psuedo-UTP; 1-propynyl-psuedouridine; 1-p-tolyl-psuedo-UTP; 1-tert-butyl-psuedo-UTP; 1-thiomethoxymethyl-psuedouridine TP; 1-thiomorpholinylmethyl-psuedouridine TP; 1-trifluoroacetyl-psuedouridine TP; 1-trifluoromethyl-psuedo-UTP; 1-vinyl-psuedouridine TP;2,2’-anhydrouridine TP; 2’-bromodeoxyuridine TP; 2’-F-5-methyl-2’-deoxy-UTP; 2’-OMe-5-Me-UTP; 2’-OMe-pseudo-UTP; 2’-α-ethynyluridine TP; 2’-α-trifluoromethyluridine TP; 2’-β-ethynyluridine TP; 2’-β-trifluoromethyluridine TP; 2’-deoxy-2’,2’-difluorouridine TP; 2’-deoxy-2’-α-mercaptouridine TP; 2’-deoxy-2’-α-thiomethoxyuridine TP; 2’-deoxy-2’-β-aminouridine TP; 2’-deoxy-2’-β-azidouridine TP; 2’-deoxy-2’-β-bromouridine TP; 2’-deoxy-2’-β-chlorouridine TP; 2’-deoxy-2’-β-fluorouridine TP; 2’-deoxy-2’-β-iodouridine TP; 2’-deoxy-2’-β-mercaptouridine TP; 2’-deoxy-2’-β-thiomethoxyuridine TP; 2-methoxy-4-thiouridine; 2-methoxyuridine; 2’-O-methyl-5-(1-propynyl)uridine TP; 3-alkylpseudo-UTP; 4’-azidouridine TP; 4’-carbocyclic uridine TP; 4’-ethynyluridine TP; 5-(1-propynyl)arabinouridine TP; 5-(2-furyl)uridine TP; 5-cyanouridine TP; 5-dimethylaminouridine TP; 5’-homo-uridine TP; 5-iodo-2’-fluorodeoxyuridine TP; 5-phenylethynyluridine TP; 5-trideuteriomethyl-6-deuteriouridine TP; 5-trifluoromethyluridine TP; 5-vinylarabinouridine TP; 6-(2,2,2-trifluoroethyl)-pseudo-UTP; 6-(4-morpholino)-pseudo-UTP; 6-(4-thiomorpholino)-pseudo-UTP; 6-(substituted phenyl)-pseudo-UTP; 6-aminopseudo-UTP; 6-azidopseudo-UTP; 6-bromopseudo-UTP; 6-butylpseudo-UTP; 6-chloropseudo-UTP; 6-cyanopseudo-UTP; 6-dimethylaminopseudo-UTP; 6-ethoxypseudo-UTP; 6-carboxyethylpseudo-UTP; 6-ethylpseudo-UTP; 6-fluoropseudo-UTP; 6-formylpseudo-UTP; 6-hydroxyaminopseudo-UTP; 6-hydroxypseudo-UTP; 6-iodopseudo-UTP; 6-isopropylpseudo-UTP; 6-methoxypseudo-UTP; 6-methylaminopseudo-UTP; 6-methylpseudo-UTP; 6-phenylpseudo-UTP; 6-phenylpseudo-UTP; 6-propylpseudo-UTP; 6-tert-butylpseudo-UTP; 6-trifluoromethoxypseudo-UTP; 6-trifluoromethylpseudo-UTP; α-thiopseudo-UTP; pseudouridine 1-(4-methylbenzenesulfonate)TP; pseudouridine 1-(4-methylbenzoate)TP; pseudouridine TP 1-[3-(2-ethoxy)]propionate; pseudouridine TP1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionate; pseudouridine TP 1-[3-{2-(2-[2-{2-(2-ethoxy)-ethoxy)-ethoxy}-ethoxy]-ethoxy}]propionate;Pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP diethyl 1-methylphosphonate; Pseudouridine TP-N1-3-propionic acid; Pseudouridine TP-N1-4-butyric acid; Pseudouridine TP-N1-5-valeric acid; Pseudouridine TP-N1-6-caproic acid; Pseudouridine TP-N1-7-heptanoic acid; Pseudouridine TP-N1-methyl-p-benzoic acid; Pseudouridine TP-N1-p-benzoic acid; Wybutosine; Hydroxywybutosine; Isowybutosine; Peroxywybutosine; Hypomodified hydroxywybutosine; 4-Deoxymethylwyosine; 2,6-(diamino)purine; 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl:1,3-(diaz a)-2-(oxo)-phenothiazin-1-yl; 1,3-(diaz a)-2-(oxo)-phenoxazin-1-yl; 1,3,5-(triaza)-2,6-(dioxo)-naphthalene; 2(amino)purine; 2,4,5-(trimethyl)phenyl; 2'-methyl, 2'-amino, 2'-azido, 2'-fluoro-cytidine; 2'-methyl, 2'-amino, 2'-azido, 2'-fluoro-adenine; 2'-methyl, 2'-amino, 2'-azido, 2'-fluoro-uridine; 2'-amino-2'-deoxyribose; 2-amino-6-chloropurine; 2-azainosinyl; 2'-azido-2'-deoxyribose; 2'-fluoro-2'-deoxyribose; 2'-fluoro-modified base; 2'-O-methylribose; 2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidin-3-yl; 2-pyridone; 3-nitropyrrole; 3-(methyl)-7-(propargyl)isoquinolinyl; 3-(methyl)isoquinolinyl; 4-(fluoro)-6-(methyl)benzimidazole; 4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5-nitroindole; 5-substituted pyrimidine; 5-(methyl)isoquinolinyl; 5-nitroindole; 6-(aza)pyrimidine; 6-(aza)thymine; 6-(methyl)-7-(aza)indolyl; 6-chloropurine; 6-phenylpyrrolopyrimidin-2-one-3-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaz a)-2-(oxo)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaz a)-2-(oxo)-phenothiazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaz a)-2-(oxo)-phenoxazin-1-yl; 7-(aza)indolyl; 7-(guanidylalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinyl;7-(guanidylalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl; 7-(guanidylalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(guanidylalkylhydroxy)-1,3-(diaz a)-2-(oxo)-phenoxazin-1-yl; 7-(guanidylalkyl-hydroxy)-1,3-(diaz a)-2-(oxo)-phenothiazin-1-yl; 7-(guanidylalkylhydroxy)-1,3-(diaz a)-2-(oxo)-phenoxazin-1-yl; 7-(propargyl)isoquinolinone; 7-(propargyl)isoquinolinone, propargyl-7-(aza)indolyl; 7-deazainosinyl; 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted 1,3-(diaz a)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidazopyridyl; aminoindolyl; anthryl; bis-o-(aminoalkylhydroxy)-6-phenylpyrrolopyrimidin-2-one-3-yl; bis-o-substituted-6-phenylpyrrolopyrimidin-2-one-3-yl; difluorotolyl; hypoxanthine; imidazopyridyl; inosinyl; isoquinolinone; isoguanosine; N2-substituted purine; N6-methyl-2-aminopurine; N6-substituted purine; N-alkylated derivative; naphthyl; nitrobenzimidazolyl; nitroimidazolyl; nitroindazolyl; nitropyrazolyl; Nubularine; O6-substituted purine; O-alkylated derivative; o-(aminoalkylhydroxy)-6-phenylpyrrolopyrimidin-2-one-3-yl; o-substituted 6-phenylpyrrolopyrimidin-2-one-3-yl; Oxoformycin TP; p-(aminoalkylhydroxy)-6-phenylpyrrolopyrimidin-2-one-3-yl; p-substituted 6-phenylpyrrolopyrimidin-2-one-3-yl; pentacenyl; Phenanthracenyl; phenyl; propargyl-7-(aza)indolyl; pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolopyrimidin-2-one-3-yl; pyrrolopyrimidinyl; pyrrolopyrazinyl; Stilbenzyl; substituted 1,2,4-triazole; Tetracenyl; Tubercidine; xanthine; xanthosine-5'-TP; 2-thiozebularine; 5-azathiozebularine; 7-deaza-2-aminopurine; pyridin-4-one ribonucleoside; 2-aminoriboside-TP; formycin A TP; formycin B TP; Pyrrolosine TP; 2'-OH-arabinosyladenosine TP; 2'-OH-cytarabine TP; 2'-OH-arabinouridine TP; 2'-OH-arabinosylguanosine TP; 5-(2-methoxycarbonylviny l)uridine TP;or N6-(19-aminopentadecaoxanonadecyl)adenosine TP;

[0353] In some embodiments, the percentage of standard A substituted by a nucleotide modified with a substitutable A (such as those nucleotides above) is at least: 0.1%, 0.5%, 0.8%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9% or 100%. In some embodiments, the percentage of standard A substituted by m 6 A can be 0.1 - 5%, particularly 0.5 - 2%, particularly 0.8 - 1.2%, such as about 1% (or 1%); in these embodiments, the RNA can be circular RNA. The low substitution level (such as 1%) of A substituted by m 6 A has been shown to inhibit innate immune activation

[23] . In some embodiments, the percentage of standard C substituted by a nucleotide modified with a substitutable cytosine (such as those nucleotides above) is at least: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9% or 100%. In some embodiments, the percentage of standard G substituted by a nucleotide modified with a substitutable G (such as those nucleotides above) is at least: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9% or 100%. In preferred embodiments, the percentage of standard U substituted by a nucleotide modified with a substitutable U (such as those nucleotides above) is at least: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or at least 99.9%, or preferably 100%; more preferably substituted by 1mΨ and / or Ψ (even more preferably 1mΨ).

[0354] In a preferred embodiment, the one or more modified ribonucleotides detailed above are or comprise 1mΨ and / or Ψ, more preferably 1mΨ. In such embodiments, the RNA can comprise 1mΨ and / or Ψ without comprising standard U ribonucleotides or other modified U ribonucleotides (i.e., no standard U ribonucleotides or modified U ribonucleotides other than 1mΨ and / or Ψ in the RNA; i.e., 100% U substitution). In particular, the RNA can comprise 1mΨ and / or Ψ without comprising standard U ribonucleotides or other modified ribonucleotides (i.e., no standard U nucleotides or any type of modified ribonucleotides other than 1mΨ and / or Ψ (capable of substituting A, C, G, or U) in the RNA; i.e., 100% U substitution, no other modified nucleotides allowed). The RNA can comprise Ψ without comprising standard U ribonucleotides or other modified U ribonucleotides (i.e., 100% U substituted by Ψ). In particular, the RNA can comprise Ψ without comprising standard U ribonucleotides or other modified ribonucleotides (i.e., 100% U substituted by Ψ, no other modified nucleotides allowed). More preferably, the RNA comprises 1mΨ without comprising standard U ribonucleotides or other modified U ribonucleotides (i.e., 100% U substituted by 1mΨ). In an even more preferred embodiment, the RNA comprises 1mΨ without comprising standard U ribonucleotides or other modified ribonucleotides (i.e., 100% U substituted by 1mΨ, no other modified nucleotides allowed). In the embodiments of this paragraph, "[may] comprise... without comprising [X]... or [Y]" can be used interchangeably with the wording "[may] comprise... and not comprise... [X] and / or [Y]".

[0355] Preferably, the RNA is codon-optimized. Codon optimization can provide an increased GC content relative to non-codon-optimized RNA encoding the same protein(s). The GC content (percentage of all ribonucleotides that are G or C in the RNA (alternatively defined as all "nitrogenous bases")) can be at least 10%, such as at least 20%, 30%, 35% or at least 40%, preferably at least 45%, 46%, 47%, 48%, 49% or at least 50%. The GC content of the RNA can be 10 - 70%, such as 20 - 65%, 30 - 65% or 35 - 65%, preferably 40 - 60%, 45 - 55%, 46 - 53%, 47 - 51% or 48 - 50%. The GC content of the RNA can be 30 - 70%, such as 40 - 70%, 45 - 70%, 50 - 70% or 55 - 70%. Codon optimization can provide an increased C content relative to non-codon-optimized RNA encoding the same protein(s). As a result of codon optimization, the percentage of C-optimisable codons in the RNA that have been replaced with codons having a greater C content (while encoding the same amino acid) can be at least 30%, such as at least 40%, 50%, 55% or at least 60%, preferably at least 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72% or at least 72%; as a result of codon optimization, the percentage of C-optimisable codons in the RNA that have been replaced with codons having a greater C content (while encoding the same amino acid) can be 30 - 80%, such as 40 - 90%, 45 - 90%, 50 - 80%, 55 - 80% or 60 - 80%, preferably 65 - 75%, 66 - 75%, 67 - 75%, 68 - 75%, 69 - 75%, 70 - 74%, 71 - 74% or 72 - 74%.

[0356] Generally, the RNA comprises a 5' and / or 3' untranslated region (UTR), preferably both a 5' UTR and a 3' UTR; for example, the 5' UTR and 3' UTR of an RNA transcript selected from the following genes (preferably the following human genes): beta-actin, albumin, ATP synthase beta subunit, fibroblast activation protein ("FAP"), histone cluster 1 H4 family member 15 ("HIST2H4A"), glyceraldehyde-3-phosphate dehydrogenase, heat shock protein family A (Hsp70) member 8 gene, interleukin-2 gene ("IL-2") and transferrin. In some preferred embodiments, the RNA comprises a 5' UTR and a 3' UTR selected respectively from:

[0357] -SEQ ID NO:61 and 62,

[0358] -SEQ ID NO:63 and 64,

[0359] - SEQ ID NO: 65 and 66,

[0360] - SEQ ID NO: 67 and 68,

[0361] - SEQ ID NO: 69 and 70, and

[0362] - RNA sequences that are at least 70%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or at least 99.5% identical to SEQ ID NO: 61, 63, 65, 67 or 69 (for 5' UTR), and RNA sequences that are at least 70%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or at least 99.5% identical to SEQ ID NO: 62, 64, 66, 68 or 70 (for 3' UTR) (in particular, pairs of 5' UTR and 3' UTR having such identity with SEQ ID NO: 61 and 62, SEQ ID NO: 63 and 64, SEQ ID NO: 65 and 66, SEQ ID NO: 67 and 68, and SEQ ID NO: 69 and 70 respectively);

[0363] wherein the RNA sequences according to SEQ ID NO: 61 and 62, SEQ ID NO: 67 and 68, SEQ ID NO: 69 and 70 (and RNA sequences having such identity therewith, preferably at least 95% or higher) are more preferred; and the RNA sequences according to SEQ ID NO: 61 and 62 (and RNA sequences having such identity therewith, preferably at least 95% or higher) are even more preferred.

[0364] Both the 3' and 5' UTRs can affect the expression of the RSV-F protein of the present disclosure through multiple mechanisms. Without being limited to this theory, the 5' UTR can at least affect the expression of the RSV-F protein of the present disclosure, for example, through pre-initiation complex regulation, closed-loop regulation, upstream open reading frame regulation (i.e., re-initiation), providing an internal ribosome entry site, and providing a microRNA binding site. Without being limited to this theory, the 3' UTR can at least affect the expression of the RSV-F protein of the present disclosure, for example, by providing regulatory regions that affect expression post-transcriptionally; for example, affecting translation efficiency, the localization of the RNA, the stability of the RNA, polyadenylation, and the circularization of the RNA.

[0365] In a specific embodiment, the RNA is circular RNA.

[0366] In a preferred embodiment, the RNA meets any 2, 3, 4, or 5 of the following criteria (e.g., (a), (b), (d), and (f); (a), (b), (c), (d), and (f); or (a), (b), (d), (e), and (f)):

[0367] (a) is non-self-replicating;

[0368] (b) is single-stranded;

[0369] (c) contains a 5’ cap which is 7’-methylguanosine linked 5’-to-5’ by a triphosphate bridge to the 5’ first ribonucleotide, and wherein the first 5’ ribonucleotide contains 2’-O-methylated ribose (2’-O-Me);

[0370] (d) contains a 3’ polyA tail;

[0371] (e) contains 1mΨ and does not contain standard U ribonucleotides or other modified ribonucleotides;

[0372] (f) contains 5’ and 3’ UTRs.

[0373] More preferably, the RNA meets all of the above criteria (a)-(f).

[0374] Generally, the RNA will contain in the 5’ to 3’ direction: a 5’ cap, a 5’ UTR, an open reading frame encoding at least the RSV-F protein of the present disclosure, a 3’ UTR, and a 3’ polyA tail (in particular, the 5’ cap as detailed above throughout this subsection; the 5’ UTR, 3’ UTR, and 3’ polyA tail).

[0375] In a preferred embodiment, the RNA comprises or consists of the following sequence:

[0376] SEQ ID NO:71; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or at least 99.94% identical thereto, preferably encoding the RSV-F protein of the present disclosure that contains the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V, and V459M relative to SEQ ID NO:1 (and encoded according thereto);

[0377] SEQ ID NO:142; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0378] SEQ ID NO:72; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0379] SEQ ID NO:143; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0380] SEQ ID NO:73; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, S215A, N228K, A241N, K315I, S348N, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0381] SEQ ID NO:74; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, S215A, N228K, K315I, S348N, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0382] SEQ ID NO:75; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0383] SEQ ID NO:76; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0384] SEQ ID NO:77; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0385] SEQ ID NO:144; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0386] SEQ ID NO:78; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0387] SEQ ID NO:115; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0388] SEQ ID NO:116; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0389] SEQ ID NO:117; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0390] SEQ ID NO:79; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0391] SEQ ID NO:145; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0392] SEQ ID NO:118; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0393] SEQ ID NO:119; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0394] SEQ ID NO:120; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding the RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0395] SEQ ID NO:80; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding the RSV-F protein of the present disclosure that comprises substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby), or

[0396] SEQ ID NO:83; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 99.94% identical thereto, preferably encoding the RSV-F protein of the present disclosure that comprises substitution N228K relative to SEQ ID NO:1 (and encoded thereby).

[0397] In a further independent aspect, the present disclosure also provides a DNA construct (preferably a DNA plasmid) encoding an RNA sequence comprising or consisting of any one of SEQ ID NOs:71 - 80, or any of the foregoing sequences having identity with any one of SEQ ID NOs:71 - 80.

[0398] In a preferred embodiment, the RNA comprises an open reading frame (ORF) comprising or consisting of the following sequence:

[0399] positions 32 - 1753 of SEQ ID NO:71; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding the RSV-F protein of the present disclosure that comprises substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0400] Positions 32 - 1753 of SEQ ID NO:142; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0401] Positions 32 - 1753 of SEQ ID NO:72; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0402] Positions 32 - 1753 of SEQ ID NO:143; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0403] positions 32-1753 of SEQ ID NO:73; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, S215A, N228K, A241N, K315I, S348N, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0404] positions 32-1753 of SEQ ID NO:74; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, S215A, N228K, K315I, S348N, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0405] positions 32-1753 of SEQ ID NO:75; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S211N, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0406] Positions 32 - 1753 of SEQ ID NO:76; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, S211N, S215A, N228K, K315I, A346Q, S348N, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0407] Positions 32 - 1753 of SEQ ID NO:77; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0408] Positions 32 - 1753 of SEQ ID NO:144; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0409] Positions 32 - 1753 of SEQ ID NO:78; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to the said position, preferably encoding an RSV-F protein of the present disclosure that comprises the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0410] Positions 32 - 1744 of SEQ ID NO:115; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to the said position, preferably encoding an RSV-F protein of the present disclosure that comprises the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0411] Positions 32 - 1693 of SEQ ID NO:116; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to the said position, preferably encoding an RSV-F protein of the present disclosure that comprises the substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0412] positions 32 - 1678 of SEQ ID NO:117; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, K445D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0413] positions 32 - 1753 of SEQ ID NO:79; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0414] positions 32 - 1753 of SEQ ID NO:145; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0415] Positions 32 - 1744 of SEQ ID NO:118; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0416] Positions 32 - 1693 of SEQ ID NO:119; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0417] Positions 32 - 1678 of SEQ ID NO:120; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to said positions, preferably encoding an RSV-F protein of the present disclosure comprising the substitutions S55T, V152R, Q210H, S215A, N228K, A241N, K315I, A346Q, S348N, K419D, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby);

[0418] positions 32-1753 of SEQ ID NO:80; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to the positions, preferably encoding the RSV-F protein of the present disclosure comprising substitutions S55T, V152R, S215A, N228K, K315I, A346Q, S348N, T455V and V459M relative to SEQ ID NO:1 (and encoded thereby), or

[0419] positions 32-1753 of SEQ ID NO:83; or an RNA sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or preferably at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% identical to the positions, preferably encoding the RSV-F protein of the present disclosure comprising substitution N228K relative to SEQ ID NO:1 (and encoded thereby).

[0420] In a further independent aspect, the present disclosure also provides a DNA construct (preferably a DNA plasmid) encoding an RNA sequence comprising an ORF; the ORF comprising or consisting of the following sequences: positions 32-1753 of any one of SEQ ID NO:71-80, or any of the foregoing sequences having identity with positions 32-1753 of any one of SEQ ID NO:71-80.

[0421] Nucleic acid (e.g., RNA) alignments can be performed, for example, visually or by any well-known algorithm; for example, using the NCBI BLAST algorithm, such as "megablast", for example, using default settings (e.g., available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&BLAST_SPEC=GeoBlast&PAGE_TYPE=BlastSearch); or for example, using the "Muscle" algorithm (see, e.g.,

[24] ,

[25] ), for example, using default settings; wherein the Muscle algorithm is preferred. The corresponding nucleotide or ribonucleotide positions are readily identifiable to the person skilled in the art and can be identified by aligning the nucleotide or ribonucleotide sequences using any well-known method (e.g., visually or algorithmically, such as the methods detailed above).

[0422] The RNA can be conveniently prepared by in vitro transcription (IVT). The IVT can use a (DNA) template that is generated and propagated in bacteria in the form of a plasmid, or is generated synthetically (e.g., by gene synthesis and / or polymerase chain reaction (PCR) engineering methods). For example, a DNA-dependent RNA polymerase (such as phage T7, T3, or SP6 RNA polymerase) can be used to transcribe the replicated RNA from the DNA template. Appropriate capping and polyA addition reactions can be used as needed (although the polyA tail is usually encoded within the DNA template).

[0423] Vector containing nucleic acid encoding RSV-F protein in pre-fusion conformation

[0424] Nucleic acids (especially RNA) are not protected per se, may be degraded by the subject's nucleases, and may require a vector to facilitate entry into target cells. Accordingly, the present disclosure also provides a vector that comprises a nucleic acid (preferably RNA) encoding the RSV-F protein of the present disclosure. The vector can be lipid-based (e.g., lipid nanoparticles or cationic nanoemulsions), polymer-based (e.g., comprising polyamines, dendrimers, and / or copolymers), peptide- or protein-based (e.g., comprising protamine, cationic cell-penetrating peptides, and / or anionic peptides conjugated to a positively charged polymer), cell-based (e.g., antigen-presenting cells such as dendritic cells loaded with the nucleic acid), or virus-based (e.g., virus replicon particles). In a specific embodiment, the vector is non-viral particle, i.e., free of or substantially free of a viral capsid.

[0425] In particular, lipid-based vectors provide a means to protect the nucleic acid (preferably RNA), e.g., by encapsulation, and deliver it to target cells for protein expression. In certain embodiments, the lipid-based vector is or comprises a cationic nanoemulsion ("CNE"). CNE and methods for their preparation are described, for example, in

[26] . For CNE, the nucleic acid (preferably RNA) encoding the RSV-F protein of the present disclosure is complexed with CNE particles, particularly comprising an oil core and a cationic lipid. The cationic lipid can interact with the negatively charged molecule, thereby anchoring the molecule to the emulsion particle. In a specific embodiment, the lipid-based vector is a lipid inorganic nanoparticle ("LION").

[0426] LNP

[0427] In a preferred embodiment, the nucleic acid (preferably RNA) is encapsulated in a lipid nanoparticle (LNP). Accordingly, in a preferred embodiment, the present invention also provides an LNP that encapsulates a nucleic acid (preferably RNA) encoding the RSV-F protein of the present invention.

[0428] Multiple such LNPs will be part of a composition (e.g., a pharmaceutical composition as detailed in the section titled "Pharmaceutical Compositions" below) comprising free and / or encapsulated nucleic acid (preferably RNA), and in some embodiments, these LNPs encapsulate at least: 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or at least 100% of the total number of nucleic acid (preferably RNA) molecules in the composition. The diameter of at least 80% of these LNPs in the composition can be 20 - 200 nm, 40 - 190 nm, 60 - 180 nm, or particularly 80 - 160 nm. In specific embodiments, the diameter of substantially all or all of the LNPs in the composition is 20 - 200 nm, 40 - 190 nm, 60 - 180 nm, or particularly 80 - 160 nm.

[0429] The LNP can comprise multilamellar vesicles (MLV), small unilamellar vesicles (SUV) or large unilamellar vesicles (LUV).

[0430] The amount of nucleic acid (preferably RNA) per LNP can vary, and the number of individual nucleic acid molecules per LNP can depend on the characteristics of the particles used. For RNA molecules, generally, an LNP can contain 1 - 500 RNA molecules, such as <200, <100, <50, <20, <10, <5 or 1 - 4. Generally, an LNP contains fewer than 10 different RNA species, such as fewer than 5, 4, 3 or 2 different species. Preferably, the LNP contains a single RNA species (i.e., all RNA molecules in the particle have the same sequence).

[0431] The LNPs according to the present disclosure can be formed from a single lipid (e.g., a cationic lipid), or particularly, from a mixture of lipids. In particular, the mixture comprises various classes of lipids, such as:

[0432] (a) A mixture of a cationic lipid and a sterol,

[0433] (b) A mixture of a cationic lipid and a neutral lipid,

[0434] (c) A mixture of a cationic lipid and a polymer-conjugated lipid,

[0435] (d) A mixture of a cationic lipid, a sterol and a polymer-conjugated lipid, or

[0436] (e) A mixture of a cationic lipid, a neutral lipid and a polymer-conjugated lipid;

[0437] Or preferably:

[0438] (f) A mixture of a cationic lipid, a sterol and a neutral lipid;

[0439] Or more preferably:

[0440] (g) A mixture of a cationic lipid, a neutral lipid, a sterol and a polymer-conjugated lipid.

[0441] Other classes of lipids, such as anionic lipids, may also be present in the mixture of lipids.

[0442] The pKa of the cationic lipid can be 5.0 - 10.0, 5.0 - 9.0, 5.0 - 8.5, preferably 5.0 - 8.0, 5.0 - 7.9 or 5.0 - 7.8, 5.0 - 7.7, or more preferably 5.0 - 7.6. The pKa of the cationic lipid is different from the pKa of the entire LNP (sometimes referred to as the "apparent pKa"). The pKa can be determined by any well-known method, such as by fluorescence measurement with 2-(p-toluenesulfonamido)-naphthalene-6-sulfonic acid (TNS) or acid-base titration; preferably by TNS fluorescence measurement; more preferably according to Example 8.

[0443] The cationic lipid preferably comprises a tertiary amine or quaternary amine group, more preferably a tertiary amine group. Exemplary cationic lipids comprising a tertiary amine group include: 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinolenylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linolenyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinolenyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinolenyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ), 3-(N,N-dilinolenylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinolenyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinolenyl-4-dimethylaminomethyl[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinolenyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane (DLin-KC2-DMA), dilinolenylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA); or MC3 (see, e.g.,

[27] ).

[0444] In some embodiments, the cationic lipid has the structure of the following lipids: RV28, RV31, RV33, RV37, RV39, RV42, RV44, RV73, RV75, RV81, RV84, RV85, RV86, RV88, RV91, RV92, RV93, RV94, RV95, RV96, RV97, RV99 or RV101, as disclosed in

[28] . In further embodiments, the cationic lipid has the following structure:

[0445]

[0446] In preferred embodiments, the cationic lipid has the following structure:

[0447]

[0448] (also known as lipid RV39).

[0449] In another preferred embodiment, the cationic lipid has the following structure:

[0450]

[0451] In another preferred embodiment, the cationic lipid has the following structure:

[0452]

[0453] The lipids in the LNP can comprise (in mol%) 20 - 80, 25 - 75, 30 - 70 or 35 - 65%, preferably 30 - 60, 40 - 55 or 40 - 50% cationic lipid; about 40% (or 40%), about 42% (or 42%), about 44% (or 44%), about 46% (or 46%) or about 48% (or 48%) cationic lipid. The lipids in the LNP can comprise (in mol%) at least 20, 25 or at least 35%, or preferably at least 40% cationic lipid. The lipids in the LNP can comprise (in mol%) not more than 80, 70 or not more than 60% or preferably not more than 50% cationic lipid.

[0454] The molar ratio of the protonatable nitrogen atoms in the cationic lipid of the LNP to the phosphate in the nucleic acid (preferably RNA), also known as the "N:P" ratio, can be in the following ranges (including the endpoints): in the range of 1:1 - 20:1, 2:1 - 10:1, 3:1 - 9:1 or 4:1 - 8:1; preferably 4.5:1 - 7.5:1, 4.5:1 - 6.5:1 or 5.0:1 - 6.5:1.

[0455] The polymer-conjugated lipid is preferably a PEGylated lipid. In the LNP, the average molecular weight of the PEG of such PEGylated lipid can be 0.5 - 11.0 kDa; such as 0.5 - 8.0, 0.8 - 8.0, 0.8 - 7.0, 0.8 - 6.0, 0.8 - 5.0, 0.8 - 4.0, 1.0 - 4.0 or 1.0 - 3.5 kDa, preferably 1.0 - 3.0, 1.2 - 2.8, 1.4 - 2.6, 1.5 - 2.5, 1.6 - 2.4 or 1.7 - 2.3 kDa, or more preferably 1.8 - 2.2, 1.9 - 2.1, about 2.0 (or 2.0 kDa). The average molecular weight of such PEG can be expressed as the median molecular weight. In the LNP, the weight-average molecular weight of the PEG of such PEGylated lipid can be 0.5 - 11.0 kDa; such as 0.5 - 8.0, 0.8 - 8.0, 0.8 - 7.0, 0.8 - 6.0, 0.8 - 5.0, 0.8 - 4.0, 1.0 - 4.0 or 1.0 - 3.5 kDa, preferably 1.0 - 3.0, 1.2 - 2.8, 1.4 - 2.6, 1.5 - 2.5, 1.6 - 2.4 or 1.7 - 2.3 kDa, or more preferably 1.8 - 2.2, 1.9 - 2.1, about 2.0 (or 2.0 kDa). Alternatively, in the LNP, the number-average molecular weight of the PEG of such PEGylated lipid can be 0.5 - 11.0 kDa; such as 0.5 - 8.0, 0.8 - 8.0, 0.8 - 7.0, 0.8 - 6.0, 0.8 - 5.0, 0.8 - 4.0, 1.0 - 4.0 or 1.0 - 3.5 kDa, preferably 1.0 - 3.0, 1.2 - 2.8, 1.4 - 2.6, 1.5 - 2.5, 1.6 - 2.4 or 1.7 - 2.3 kDa, or more preferably 1.8 - 2.2, 1.9 - 2.1, about 2.0 (or 2.0 kDa). Alternatively, in the LNP, the molecular weight of at least 80% of the PEG of such PEGylated lipid can be 0.5 - 11.0 kDa; such as 0.5 - 8.0, 0.8 - 8.0, 0.8 - 7.0, 0.8 - 6.0, 0.8 - 5.0, 0.8 - 4.0, 1.0 - 4.0 or 1.0 - 3.5 kDa, preferably 1.0 - 3.0, 1.2 - 2.8, 1.4 - 2.6, 1.5 - 2.5, 1.6 - 2.4 or 1.7 - 2.3 kDa, or more preferably 1.8 - 2.2, 1.9 - 2.1, about 2.0 or 2.0 kDa.

[0456] The PEGylated lipid can have the following structure:

[0457]

[0458] Exemplary PEGylated lipids include 2-[(polyethylene glycol)-2000]-N,N-ditetradecylethanamide and 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000, 1,2-dimyristoyl-sn-glycero-2-phosphoethanolamine-N-[methoxy(polyethylene glycol)], and 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol. Preferably, the PEGylated lipid is 2-[(polyethylene glycol)-2000]-N,N-ditetradecylethanamide or 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000.

[0459] The lipids in the LNP can comprise (in mol %) 0.1 - 8.0, 0.4 - 7.0, 0.6 - 6.0, 0.8 - 4.0, or 0.8 - 3.5%, preferably 1.0 - 3.0% polymer-conjugated lipid (preferably PEGylated lipid); about 1.0 (or 1.0%), about 1.5% (or 1.5%), about 2.0% (or 2.0%), or about 2.5% (or 2.5%) polymer-conjugated lipid (preferably PEGylated lipid). The lipids in the LNP can comprise (in mol %) at least 0.1, 0.5, or at least 0.8%, or preferably at least 1% polymer-conjugated lipid (preferably PEGylated lipid). The lipids in the LNP can comprise (in mol %) no more than 8.0%, 6.0%, or 4.0%, or preferably no more than 3.0% polymer-conjugated lipid (preferably PEGylated lipid).

[0460] Preferably, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), although other neutral lipids available to those skilled in the art can also be used.

[0461] The lipids in the LNP can comprise (in mol %) 0 - 15.0, 0.1 - 15.0, 2.0 - 14.0, 5.0 - 13.0, 6.0 - 12.0, or 7.0 - 11.0%, preferably 8.0 - 11.0% or 9.0 - 11.0% neutral lipid; about 9.4% (or 9.4%), about 9.6% (or 9.6%), about 9.8% (or 9.8%), or about 10.0% (or 10%) neutral lipid. The lipids in the LNP can comprise (in mol %) at least 0.1, 5.0, or at least 7.0%, or preferably at least 8.0% or at least 9.0% neutral lipid. The lipids in the LNP can comprise (in mol %) no more than 15.0, 13.0, or no more than 12.0%, or preferably no more than 11.0% neutral lipid.

[0462] Exemplary sterols include cholesterol, cholesteryl sulfate, desmosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, dihydrolanosterol, coprosterol, campesterol, 14-demethyl lanosterol, 8(9)-dehydrocholesterol, 8(14)-dehydrocholesterol, 14-demethyl-14-dehydro lanosterol (FF-MAS), diosgenin, dehydroepiandrosterone sulfate (DHEA sulfate), dehydroepiandrosterone, sitosterol, lanosterol-95, 4,4-dimethyl(d6)-cholest-8(9),14-dien-3β-ol (dihydro-FF-MAS-d6), 4,4-dimethyl(d6)-cholest-8(9)-en-3β-ol (dihydro T-MAS-d6), ergosterol, sitostanol, campestanol, campestanol, 7-dehydrodesmosterol, pregnenolone, 4,4-dimethyl-cholest-8(9)-en-3β-ol (dihydro T-MAS), Δ5-avenasterol, brassicasterol, dihydro FF-MAS, 24-methylene cholesterol, oxysterol, deuterated sterol, fluorinated sterol, sulfonated sterol, phosphorylated sterol, A-ring substituted sterol, cholest-5-ene-3β,4β-diol, 5α-cholestan-3β-ol, 4-cholesten-3-one, cholest-8(9),24-dien-3-one, cholest-8(9),24-dien-3-one, 2,2,3,4,4-pentadeuterio-5α-cholestan-3β-ol, cholesteryl phosphocholine, cholesterol-d7 pentadecanoate, cholesterol-d7 palmitate, B-ring substituted sterol, cholestanol, 5β,6β-epoxy-d7, 3β-hydroxy-5-cholesten-7-one, 6α-hydroxy-5α-cholestane, cholestanol, 5α,6α-epoxy, cholest-5-ene-3β,7α-diol, cholest-5-ene-3β,7β-diol, cholestanol, 5α,6α-epoxy-d7, Δ5,7-cholestadiene, cholest-5,8(9)-dien-3β-ol, cholest-5,8(14)-dien-3β-ol, 7α-hydroxy-4-cholesten-3-one, ergosterol-d7, ergosterol, 7-dehydrodesmosterol, 3β,5α-dihydroxy-cholestan-6-one, D-ring substituted sterol, 3β-hydroxy-5α-cholest-8(14)-en-15-one, 3β-hydroxy-5α-cholestan-15-one, 5α-cholest-8(14)-ene-3β,15α-diol, 5α-cholest-8(14)-ene-3β,15β-diol, lanosterol-95, 5α-7,24-cholestadiene, 14-dehydroergosterol, ergosta-5,7,9(11),22-tetraen-3β-ol, cholest-5-ene-3β,25-diol, cholest-(25R)-5-ene-3β,27-diol, 24(R / S),25-epoxycholesterol, 24(S),25-epoxycholesterol, 24(R / S),25-epoxycholesterol-d6, cholest-5-ene-3β,22(S)-diol, cholest-5-ene-3β,22(R)-diol, cholest-5-ene-3β,24(S)-diol, cholest-5-ene-3β,24(R)-diol, 27-hydroxy-4-cholesten-3-one, campestanol, N,N-dimethyl-3β-hydroxy cholenamide, 25,27-dihydroxycholesterol, N,N-dimethyl-3β-hydroxy cholenamide, 25,27-dihydroxycholesterol, 5-cholestene-3β,20α-diol, 24S,25-epoxy-5α-cholest-8(9)-en-3β-ol, 24(S / R),25-epoxy lanost-8(9)-en-3β-ol, 7-keto-27-hydroxycholesterol, 7α,27-dihydroxy-4-cholesten-3-one, 7α,27-dihydroxycholesterol, 7β,27-dihydroxycholesterol, 5α,6β-dihydroxy campestanol, 7α,25-dihydroxycholesterol, 7β,25-dihydroxycholesterol, 7α,24(S)-dihydroxycholesterol, 7α,24(S)-dihydroxy-4-cholesten-3-one, 7-keto-25-hydroxycholesterol, 7α,24S,27-trihydroxycholesterol, dihydrotestosterone, testosterone, estrone, estrogen, estradiol, corticosterone, cortisol or 24S,27-dihydroxycholesterol.,

[0463] Preferably, the sterol is cholesterol or a cholesterol-based lipid (such as any of the lipids provided in the preceding paragraph).

[0464] The lipids in the LNP may comprise (in mol%) 20 - 80, 25 - 80, 30 - 70, 30 - 60, 35 - 60 or 40 - 60%, preferably 40 - 50% or 41 - 49% sterol; about 42% (or 42%), about 43% (or 43%), about 44% (or 44%), about 46% (or 46%) or about 48% (or 48%) sterol. The lipids in the LNP may comprise (in mol%) at least 20, 30 or at least 35%, or preferably at least 40% or at least 41% sterol. The lipids in the LNP may comprise (in mol%) no more than 80, 70 or no more than 60%, or preferably no more than 50% sterol.

[0465] The lipids in the LNP may have the following molar% combinations: 30 - 60% cationic lipid (such as 35 - 55%, or preferably 40 - 50%), 35 - 70% sterol (such as 40 - 55%, or preferably 41 - 49%), 0.8 - 4.0% polymer-conjugated lipid (such as 0.8 - 3.5%, or preferably 1.0 - 3.0%) and 0 - 15% neutral lipid (such as 6.0 - 12.0% or preferably 8.0 - 11.0%).

[0466] Such LNPs encapsulating nucleic acids (preferably RNA) can be formed by admixing a first solution containing the nucleic acid with a second solution containing the lipids forming the LNP. The admixing can be carried out by any suitable method available to the person skilled in the art, such as a T-mixer, a microfluidic or an impinging jet mixer. Filtration can be carried out after admixing to obtain a desired LNP size distribution (e.g., those detailed above in this subsection). The filtration can be carried out by any suitable method available to the person skilled in the art, such as tangential flow filtration or cross-flow filtration.

[0467] According to a further independent aspect, the present disclosure provides a method for preparing an LNP encapsulating a nucleic acid (preferably RNA) encoding the RSV-F protein of the present disclosure, comprising admixing a first solution containing the nucleic acid and a second solution containing the lipids forming the LNP (e.g., using the method described in the foregoing paragraph); and optionally filtering the obtained admixture (e.g., using the method described in the foregoing paragraph).

[0468] Pharmaceutical composition

[0469] In a further independent aspect, the present disclosure also provides a pharmaceutical composition comprising the RSV-F protein, nucleic acid (preferably RNA) and / or carrier (preferably lipid nanoparticle) of the present disclosure. Such compositions generally further comprise a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients are well known in the art, see, for example,

[29] . Such compositions are generally used to immunize a subject against a disease, preferably against RSV. Thus, the pharmaceutical compositions of the present disclosure are generally regarded as vaccine compositions.

[0470] The pharmaceutical compositions of the present disclosure can contain the RSV-F protein, nucleic acid (preferably RNA) and / or carrier (preferably lipid nanoparticle) in pure water (e.g., "w.f.i") or in a buffer (e.g., phosphate buffer, Tris buffer, borate buffer, succinate buffer, histidine buffer or citrate buffer). The buffer salts will generally be included in the range of 5 - 20 mM.

[0471] The pH value of the pharmaceutical compositions of the present disclosure can be between 5.0 and 9.5, for example between 6.0 and 8.0.

[0472] The pharmaceutical compositions of the present disclosure can contain sodium salts (e.g., sodium chloride) to provide tonicity. A concentration of 10 ± 2 mg / mL NaCl is typical, for example about 9 mg / mL (or 9 mg / mL).

[0473] The pharmaceutical compositions of the present disclosure may comprise metal ion chelators (in particular, in embodiments where such compositions comprise RNA). These chelators can extend RNA stability by removing ions that can accelerate phosphodiester hydrolysis. Accordingly, such compositions may comprise one or more of the following: EDTA, EGTA, BAPTA, pentetic acid, etc. Such chelators are typically present at 10 - 500 μM (e.g., 0.1 mM). Citrates (such as sodium citrate) can also act as chelators and advantageously also provide buffering activity.

[0474] The osmolality of the pharmaceutical compositions of the present disclosure can be between 200 mOsm / kg and 400 mOsm / kg, such as between 240 - 360 mOsm / kg, or between 290 - 310 mOsm / kg.

[0475] The pharmaceutical compositions of the present disclosure may comprise one or more preservatives, such as thimerosal or 2 - phenoxyethanol. Mercury - free compositions are preferred, and vaccines can be prepared without preservatives.

[0476] The pharmaceutical compositions of the present disclosure can be aseptic or sterile.

[0477] The pharmaceutical compositions of the present disclosure can be pyrogen - free, for example containing < 1 EU (endotoxin unit, a standard measure) per dose, and preferably < 0.1 EU per dose.

[0478] The pharmaceutical compositions of the present disclosure can be gluten - free.

[0479] The pharmaceutical compositions of the present disclosure can be prepared in unit - dose form. In some embodiments, the volume of a unit dose can be between 0.1 - 1.0 mL, such as about 0.5 mL (or 0.5 mL).

[0480] The pharmaceutical compositions of the present disclosure can be prepared as injectable formulations, as solutions or suspensions. The composition can be prepared for pulmonary administration, for example by an inhaler, using a fine spray. The composition can be prepared for nasal, ear, or eye administration, for example as a spray or drops. Injectable formulations for intramuscular injection are typical.

[0481] The pharmaceutical composition of the present disclosure comprises an immunologically effective amount of RSV-F protein, nucleic acid (preferably RNA) and / or a carrier (preferably a lipid nanoparticle), and any other desired components. "Immunologically effective amount" means that administering this amount to an individual (administered as a single dose or as part of a series) is effective for treatment or prevention (preferably prevention of RSV). This amount varies depending on the health and physical condition of the individual to be treated, age, taxonomic group of the individual to be treated (such as non-human primates, primates, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection required, the formulation of the vaccine, the treating physician's assessment of the medical condition, and other relevant factors. It is expected that this amount will fall within a relatively wide range that can be determined by routine testing. In embodiments where the pharmaceutical composition of the present disclosure comprises RNA, the RNA content will typically be expressed as the amount of RNA per dose. Preferred doses have ≤120 μg RNA, for example ≤100 μg (such as 10 - 120 μg or 10 - 100 μg, e.g., 10 μg, 25 μg, 50 μg, 75 μg or 100 μg, or about 10 μg, 25 μg, 50 μg, 75 μg or 100 μg), but expression can be seen at much lower levels, such as ≤1 μg / dose, ≤100 μg / dose, ≤10 μg / dose, ≤1 μg / dose, etc.

[0482] The pharmaceutical composition of the present disclosure may further comprise an adjuvant (i.e., a reagent that enhances the immune response in a non-specific manner), particularly, but not limited to, when it comprises the RSV-F protein of the present disclosure. Common adjuvants include: suspensions of minerals (such as alum, aluminum hydroxide, aluminum phosphate) that can adsorb RSV-F protein; emulsions, including water-in-oil and oil-in-water (and their variants, including double emulsions and reversible emulsions); liposaccharide, lipopolysaccharide, immunostimulatory nucleic acids (such as CpG oligonucleotides), liposomes, Toll receptor agonists (particularly, TLR2, TLR4, TLR7 / 8 and TLR9 agonists), and various combinations of such components. In some embodiments, the adjuvant is a TLR7 agonist, such as imidazoquinoline or imiquimod. In some embodiments, the adjuvant is an aluminum salt, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate. The adjuvants described herein can be used alone or in any combination, such as alum / TLR7 (also known as AS37). The pharmaceutical composition of the present disclosure may comprise a saponin as an adjuvant, such as the saponin fraction QS21 (see, for example,

[30] ). QS21 can be used in a substantially pure form, such as at least 80% pure, e.g., at least 85%, 90%, 95% or at least 98% pure. Suitable QS-21 fractions are described in

[31] .

[0483] The pharmaceutical composition of the present disclosure (preferably when comprising a lipid nanoparticle containing the nucleic acid (preferably RNA) of the present disclosure) can be lyophilized.

[0484] In some embodiments, the pharmaceutical composition of the present disclosure comprises (i) a nucleic acid (preferably RNA) encoding the RSV-F protein of the present disclosure, and (ii) other nucleic acids (preferably RNA) encoding at least one other protein. The nucleic acids of (i) and (ii) may be contained within the same vector (preferably a lipid nanoparticle) or within separate vectors (preferably lipid nanoparticles). In a preferred embodiment, the at least one other protein is an antigen; and thus may comprise or may be a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a tumor antigen, or an allergenic (i.e., derived from or originating from an allergen) antigen. The at least one other protein will generally be a pathogen antigen. The at least one other protein will generally be an antigen that is a surface polypeptide, such as a spike glycoprotein, a hemagglutinin, an adhesin, or an envelope glycoprotein. In a specific embodiment, the at least one other protein is an antigen from or derived from a virus (especially a virus that causes a respiratory disease, especially a seasonal virus that causes a respiratory disease). In embodiments in which the at least one other protein is an antigen from or derived from a virus, examples of such viruses include: coronaviruses, orthomyxoviruses, pneumoviridae, paramyxoviridae, poxviridae, picornaviruses, bunyaviruses, hepadnaviruses, filoviruses, togaviruses, flaviviruses, pestiviruses, hepadnaviruses, rhabdoviruses, caliciviridae, retroviruses, reoviruses, parvoviruses, herpesviruses, papovaviruses, and adenoviruses.

[0485] In a preferred embodiment, the at least one other protein encoded by the nucleic acid of (ii) is a paramyxoviridae protein (especially a paramyxoviridae antigen). Useful paramyxoviridae proteins (especially, antigens) can be from orthoparamyxovirus or metaparamyxovirus, especially human RSV or human metaparamyxovirus (hMPV). Useful other hMPV antigens include, for example, F, N, P, M, M2-1 and M2 antigens (especially, the F antigen). Such hMPV proteins (especially, antigens) can be from or derived from subtype A or B. In a preferred embodiment, the nucleic acid of (i) is an RNA encoding the RSV-F protein of the present disclosure, and the nucleic acid of (ii) is an RNA encoding an hMPV antigen (especially, the F antigen). In such RNA embodiments, the preferred group of patients (in which the pharmaceutical composition can be used for therapy, especially vaccination) is infants (see the section entitled "Medical Use and Methods of Treatment" below). In addition to other RSV-F antigens (i.e., having a different amino acid sequence from the RSV-F protein of the present disclosure encoded by the nucleic acid), useful other human RSV antigens encoded by the nucleic acid of (ii) include, for example, G, M1, M2-1, M2-2, P, L, N, NS1, NS2 and SH antigens. Such other human RSV proteins (especially, antigens, especially the F antigen) can be from or derived from subtype A or B, especially subtype B.

[0486] In a preferred embodiment, the at least one other protein encoded by the nucleic acid of (ii) is a coronavirus antigen. Useful coronavirus antigens can be from SARS coronavirus, especially SARS-CoV2. Useful coronavirus antigens (preferably SARS-CoV2 antigens) include spike, M, E, HE, nucleocapsid, Plpro and 3CLPro proteins, especially the spike protein. Preferably, the coronavirus antigen is the SARS-CoV2 spike protein. The SARS-CoV2 spike protein can be from any variant, such as Omicron (e.g., Omicron BA.1, BA.2, BA3, BA.4 or BA.5), Alpha, Epsilon, Eta, Theta, Kappa, Iota, Zeta, Mu, Lambda, Beta, Gamma or Delta. Preferably, the SARS-CoV2 spike protein comprises one or more mutations relative to the wild-type protein, especially mutations of one or more (e.g., two) proline residues. The one or more mutations can be introduced to stabilize the SARS-CoV2 spike protein in the pre-fusion conformation. In a preferred embodiment, the nucleic acid of (i) is an RNA encoding the RSV-F protein of the present disclosure, and the nucleic acid of (ii) is an RNA encoding a coronavirus antigen, e.g., as detailed above. In such RNA embodiments, the preferred patient group (wherein the pharmaceutical composition can be used for therapy, especially vaccination) is the elderly (see the section entitled "Medical Use and Treatment Methods" below).

[0487] In another preferred embodiment, the at least one other protein encoded by the nucleic acid of (ii) is an orthomyxovirus antigen. Useful orthomyxovirus antigens can be from influenza A, B, or C viruses. Useful orthomyxovirus antigens (especially influenza A, B, or C virus antigens) include hemagglutinin, neuraminidase, and matrix M2 protein, especially hemagglutinin. Preferably, the orthomyxovirus antigen is influenza A virus hemagglutinin. The influenza A virus hemagglutinin can be from any subtype, such as H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16. In a preferred embodiment, the nucleic acid of (i) is an RNA encoding the RSV-F protein of the present disclosure, and the nucleic acid of (ii) is an RNA encoding an orthomyxovirus antigen, for example, as detailed above. In such RNA embodiments, the preferred group of patients (wherein the pharmaceutical composition can be used for therapy, especially vaccination) is the elderly (see the section titled "Medical Use and Treatment Methods" below). In such RNA embodiments, the nucleic acid of (i) can encode the RSV-F protein of the present disclosure, the nucleic acid of (ii) can encode an orthomyxovirus antigen, for example, as detailed above, and a third nucleic acid can be present in the pharmaceutical composition, which can encode a coronavirus antigen, for example, as detailed in the foregoing paragraph above.

[0488] In a further independent aspect, the present disclosure also provides a delivery device (such as a syringe, nebulizer, atomizer, inhaler, skin patch, etc.) that contains the pharmaceutical composition of the present disclosure. This device can be used to administer the composition to a vertebrate subject.

[0489] In a further independent aspect, the present disclosure also provides a method for preparing a pharmaceutical composition, which includes formulating the RSV-F protein, nucleic acid (preferably RNA), or carrier (preferably lipid nanoparticle) of the present disclosure with a pharmaceutically acceptable excipient to produce the composition. In particular, the pharmaceutical composition has the characteristics as detailed throughout this section above.

[0490] In a further independent aspect, the present disclosure also provides a kit that contains the RSV-F protein, nucleic acid, carrier, pharmaceutical composition, or delivery device of the present disclosure, as well as instructions for use.

[0491] Medical use and treatment method

[0492] In a further independent aspect, the present disclosure also provides the RSV-F protein, nucleic acid (preferably RNA), carrier (preferably lipid nanoparticle), or pharmaceutical composition of the present disclosure for medical use. The use will generally be in a method for enhancing the immune response of a subject.

[0493] In a further independent aspect, the present disclosure also provides the use of the RSV-F protein, nucleic acid (preferably RNA), vector (preferably lipid nanoparticle), or pharmaceutical composition of the present disclosure in the manufacture of a medicament. The medicament will generally be used to enhance the immune response of a subject.

[0494] In a further independent aspect, the present disclosure also provides a method of treatment comprising the step of administering to a subject (preferably a subject in need thereof) an effective amount of the RSV-F protein, nucleic acid (preferably RNA), vector (preferably lipid nanoparticle), or pharmaceutical composition of the present disclosure. The method will generally be used to enhance the immune response of a subject.

[0495] The immune response is preferably protective and preferably involves antibody and / or cell-mediated immunity. Generally, the subject is a vertebrate, preferably a mammal, more preferably a human or a large veterinary mammal (such as a horse, cow, deer, goat, pig), and even more preferably a human.

[0496] The RSV-F protein, nucleic acid, vector, or pharmaceutical composition of the present disclosure can be used to prevent, alleviate, or treat an infection or a disease. Additionally or alternatively, the RSV-F protein, nucleic acid, vector, or pharmaceutical composition of the present disclosure can be used to prevent, alleviate, or treat symptoms associated with an infection or a disease. The infection is generally an infection caused by a virus of the family Pneumoviridae, and the disease is generally a disease associated with a virus of the family Pneumoviridae. In a preferred embodiment, the virus of the family Pneumoviridae is a orthopneumovirus, more preferably RSV, and even more preferably human RSV (including both its A and B subtypes).

[0497] Accordingly, the present disclosure also provides the RSV-F protein, nucleic acid, vector, or pharmaceutical composition of the present disclosure; for treating or preventing RSV (preferably a method of vaccinating against RSV). The present disclosure also provides the use of the RSV-F protein, nucleic acid, vector, or pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating or preventing RSV (preferably wherein the medicament is a vaccine). The present disclosure also provides a method of inducing an immune response against RSV in a subject (preferably a method of vaccinating a subject against RSV) comprising administering to the subject an immunologically effective amount of the RSV-F protein, nucleic acid, vector, or pharmaceutical composition of the present disclosure.

[0498] Vaccination according to the present disclosure can be prophylactic (i.e., preventing infection) or therapeutic (i.e., treating infection), but will typically be prophylactic. Such vaccination methods can include single-dose administration. Alternatively, such vaccination methods can include vaccination regimens (i.e., administering multiple doses). Such regimens can involve repeated administration of immunologically identical protein antigens (in the form of or delivered by the RSV-F protein, nucleic acid, vector, or pharmaceutical composition of the present disclosure), particularly in an initial prime-boost regimen. In a prime-boost regimen, the first administration ("prime") can induce the proliferation and maturation of B and / or T cell precursors specific for one or more immunogenic epitopes present on the delivered antigen (induction phase). The second (and in some cases subsequent) administration ("boost") can further stimulate and potentially select the cellular recall response initiated by the previous administration(s). The different administrations can be given by the same or different routes, such as parenteral prime and mucosal boost, mucosal prime and parenteral boost, etc. The (one or more) prime administrations and the (one or more) boost administrations will be temporally separated, for example, by at least: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 months or more. In some embodiments, two prime administrations can be administered at intervals of 3-9 weeks (e.g., intervals of 4-9, 5-9, 6-9, 7-9, or 7-8 weeks, or an interval of about two months), followed by one or more boost administrations 4-14 months (e.g., 5-13, 6-13, 7-13, 8-13, 9-13, 10-13, or 11-13 months, or about one year) after the second prime administration. In some embodiments, the prime administration is administered to an initial subject. In some embodiments, the protein antigen can be delivered in different formats or by different formats in the prime and boost administrations. For example, the protein antigen can be delivered as a protein for the (one or more) prime administrations, and delivered by a nucleic acid (particularly RNA, particularly by a vector comprising RNA) for the (one or more) boost administrations, and vice versa. Alternatively, different nucleic acid formats can be used, such as the protein antigen can be delivered by RNA (particularly by a vector comprising RNA) for the (one or more) prime administrations, and also delivered by a viral vector (e.g., an adenovirus vector) for the (one or more) boost administrations, and vice versa.

[0499] The RSV-F protein, nucleic acid, vector or pharmaceutical composition of the present disclosure will generally be administered directly to a subject. Direct delivery can be accomplished by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, intradermal or injection into the interstitial space of tissue). Alternative delivery routes include rectal, oral (e.g., tablets, sprays), buccal, sublingual, vaginal, topical, transdermal or transcutaneous, intranasal, ocular, otic, pulmonary or other mucosal administration. Preferably, the RSV-F protein, nucleic acid, vector or pharmaceutical composition of the present disclosure will be administered intramuscularly or intradermally (especially by needle, such as a subcutaneous injection needle), more preferably intramuscularly.

[0500] The RSV-F protein, nucleic acid, lipid vector or pharmaceutical composition of the present disclosure can be used to elicit systemic and / or mucosal immunity.

[0501] The subject of the vaccination method according to the present disclosure can be a child (preferably an infant) or an adult (preferably an elderly person or a pregnant woman). Immunocompromised individuals can also be subjects of such vaccination (whether children or adults).

[0502] Infant vaccination

[0503] In a preferred embodiment, the RSV-F protein, nucleic acid, vector or pharmaceutical composition of the present disclosure is administered to an infant (preferably a human infant) as a subject of vaccination. The immune system of an infant is immature (see, e.g.,

[32] ), and thus this group is vulnerable to RSV infection and resulting diseases. Infant vaccination can prevent lower respiratory tract infections (especially bronchiolitis and (bronchial) pneumonia).

[0504] The infant can be less than one year old, such as less than: 11, 10, 9, 8, 7, 6, 5, 4 or less than 3 months old. The infant can be ≥ one month old, such as ≥: 2, 3, 4, 5 or ≥ 6 months old. Preferably, the infant is 2 - 6 months old (i.e., within 2 and 6 months old, and including 2 and 6 months old), more preferably 2 - 4 months old.

[0505] In a preferred embodiment, the infant is born to a female who has been administered an RSV vaccine (such as the RSV-F protein, nucleic acid, vector or pharmaceutical composition of the present disclosure) (preferably during pregnancy of the infant). In addition to the active immunity generated by the infant, the combination of maternal and infant vaccination can also advantageously provide passive transfer of maternal antibodies to the infant (i.e., through the placenta and / or breast milk).

[0506] Elderly vaccination

[0507] In another preferred embodiment, the RSV-F protein, nucleic acid, vector or pharmaceutical composition of the present disclosure is administered to the elderly (preferably human elderly) as a subject for vaccination. The elderly may suffer from age-related immunosenescence (reviewed in, for example,

[33] ), and thus this group of people is also vulnerable to RSV infection and resulting diseases. Vaccination of the elderly can prevent lower respiratory tract infections (in particular, pneumonia).

[0508] The elderly can be ≥50 years old, such as ≥: 55, 60, 65, 70, 75, 80, 85, 90, 95 or ≥100 years old. Preferably, the elderly are ≥60 or ≥65 years old (such as 60 - 120 or 65 - 120 years old).

[0509] Pregnant women vaccination

[0510] In another preferred embodiment, the RSV-F protein, nucleic acid, vector or pharmaceutical composition of the present disclosure is administered to pregnant women (preferably pregnant human women) as a subject for vaccination. The main purpose of maternal vaccination is to protect the infant from RSV infection at birth, for example, through the passive transfer of antibodies across the placenta and / or in breast milk.

[0511] The pregnant woman can be in the first, second or third trimester of her pregnancy, preferably the third trimester. The pregnant woman can be ≥20 weeks pregnant, such as ≥: 22, 24, 26, 28, 30, 32, 34, 36 or ≥38 weeks pregnant. Preferably, the pregnant woman is ≥28, ≥29 or ≥30 weeks pregnant (such as 28 - 43, 29 - 43 or 30 - 43 weeks pregnant).

[0512] Generally

[0513] Unless otherwise stated, the practice of the present disclosure will employ conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology.

[0514] Unless the context clearly indicates otherwise, the singular terms "a", "an" and "the" include plural referents. Similarly, unless the context clearly indicates otherwise, the word "or" is intended to include "and". The term "plurality" refers to two or more. The term "at least one" refers to one or more.

[0515] Unless otherwise specified, when a numerical range is provided, it is inclusive, i.e., it includes the endpoints.

[0516] Unless otherwise indicated, the terms "at least", "not exceeding", and other such terms before a list of values may apply to all members of the said list (and not only to its first member).

[0517] The term "comprising" encompasses "including" as well as "consisting of", e.g., a composition "comprising" X may consist only of X or may contain additional things, e.g., X + Y.

[0518] The term "about" associated with a numerical value x is optional and means, e.g., x ± 10%.

[0519] The word "substantially" does not exclude "entirely", e.g., a composition "substantially free of" Y may be entirely free of Y. Where necessary, the word "substantially" may be omitted from the definitions of the present disclosure.

[0520] References to charges, cations, anions, etc. are taken at pH 7.

[0521] Embodiment

[0522] The present disclosure also provides the following numbered embodiments. The combinations of features of the present disclosure presented below are exemplary and should not be construed as exhaustive.

[0523] 1. An RSV-F protein in a pre-fusion conformation, which is mutated relative to the wild-type RSV-F according to SEQ ID NO:1; wherein the RSV-F protein comprises at least one mutation relative to the wild-type in the region corresponding to positions 217 - 239 of SEQ ID NO:1; wherein the at least one mutation is introduced by substitution or insertion of a residue containing an H-bond donor and / or acceptor moiety in the side chain.

[0524] 2. The RSV-F protein according to embodiment 1, wherein the region corresponding to positions 217 - 239 of SEQ ID NO:1 comprises an α-helix.

[0525] 3. The RSV-F protein according to embodiment 1 or 2, wherein the region corresponding to positions 217 - 239 of SEQ ID NO:1 has at least 85%, 90% or 95% sequence identity with positions 217 - 239 of SEQ ID NO:1.

[0526] 4. The RSV-F protein according to any one of the preceding embodiments, wherein the at least one mutation introduces, by substitution or insertion, a residue selected from K, R, W, N, Q, H, S, T and Y; optionally K, R, Q and N; optionally K and R; further optionally K into the region corresponding to positions 217 - 239 of SEQ ID NO:1.

[0527] 5. The RSV-F protein according to Embodiment 4, wherein the at least one mutation introduces a residue selected from K, R, W, N, Q, H, S, T, and Y; optionally K, R, Q, and N; optionally K and R; further optionally K, by substitution or insertion into the region corresponding to positions 220-235 of SEQ ID NO:1; optionally positions 227-232; further optionally positions 228-232.

[0528] 6. The RSV-F protein according to Embodiment 5, which comprises

[0529] a substitution at position 228 of SEQ ID NO:1 with K, R, Q, or N; optionally K, R, or Q; optionally K or R; further optionally K;

[0530] and / or

[0531] a substitution at position 232 of SEQ ID NO:1 with N.

[0532] 7. The RSV-F protein according to Embodiment 6, which comprises a substitution at position 228 (N) of SEQ ID NO:1 with K or R.

[0533] 8. The RSV-F protein according to any of Embodiment 7, which comprises a substitution at position 228 (N) of SEQ ID NO:1 with K.

[0534] 9. An RSV-F protein in a pre-fusion conformation, which is mutated relative to the wild-type RSV-F according to SEQ ID NO:1; wherein the RSV-F protein comprises a substitution at position 228 of SEQ ID NO:1 with K or A.

[0535] 10. The RSV-F protein according to any of the foregoing embodiments, which further comprises (a):

[0536] (ai) at least one mutation relative to the wild-type in the region corresponding to positions 38-60 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 38-60 of SEQ ID NO:1; and / or (aii) at least one mutation relative to the wild-type in the region corresponding to positions 296-318 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 296-318 of SEQ ID NO:1, and / or introduces a residue selected from M, F, I, and V into the region by substitution or insertion.

[0537] 11. The RSV-F protein according to any one of the foregoing embodiments, which comprises (b) at least one mutation relative to the wild type in the region corresponding to positions 208-216 of SEQ ID NO:1, wherein the at least one mutation increases the hydrophobicity of the region relative to positions 208-216 of SEQ ID NO:1, and / or introduces a P residue into the region by substitution or insertion.

[0538] 12. The RSV-F protein according to any one of the foregoing embodiments, which comprises (c) at least one mutation relative to the wild type in the region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces a glycosylation site into the region by substitution or insertion; or (d) at least one mutation relative to the wild type in the region corresponding to positions 345-352 of SEQ ID NO:1, wherein the at least one mutation introduces at least one residue selected from N, D, F, H, K, L, Q, R, T, W, and Y into the region by substitution or insertion.

[0539] 13. The RSV-F protein according to any one of embodiments 10-12, wherein the regions corresponding to positions 38-60 and 296-318 of SEQ ID NO:1 each comprise a β-sheet.

[0540] 14. The RSV-F protein according to any one of embodiments 10-13, wherein the region corresponding to positions 38-60 of SEQ ID NO:1 has at least 50%, 60%, 70%, 80%, 85%, 90%, or 95% sequence identity with positions 38-60 of SEQ ID NO:1; and / or, optionally, the region corresponding to positions 296-318 of SEQ ID NO:1 has at least 50%, 60%, 70%, 80%, 85%, 90%, or 95% sequence identity with positions 296-318 of SEQ ID NO:1.

[0541] 15. The RSV-F protein according to any one of embodiments 10-14, which comprises (a) a substitution at position 55 of SEQ ID NO:1 with T, C, V, I, or F; optionally T, C, or V; optionally a substitution with T or C.

[0542] 16. The RSV-F protein according to any one of embodiments 10-15, which comprises (a) a substitution at position 55 of SEQ ID NO:1 with T.

[0543] 17. The RSV-F protein according to any one of embodiments 10-16, wherein the region corresponding to positions 208-216 of SEQ ID NO:1 comprises a loop.

[0544] 18. The RSV-F protein according to any one of Embodiments 10-17, wherein the region corresponding to positions 38-60 of SEQ ID NO:1 has at least 50%, 60%, 75% or 85% sequence identity with positions 208-216 of SEQ ID NO:1.

[0545] 19. The RSV-F protein according to any one of Embodiments 10-18, which comprises (b) a substitution at position 215 of SEQ ID NO:1 with A, P, V, I or F; optionally a substitution with A or P.

[0546] 20. The RSV-F protein according to any one of Embodiments 10-19, which comprises (b) a substitution at position 215 of SEQ ID NO:1 with A.

[0547] 21. The RSV-F protein according to any one of Embodiments 10-20, wherein the region corresponding to positions 345-352 of SEQ ID NO:1 comprises a beta sheet and a loop.

[0548] 22. The RSV-F protein according to any one of Embodiments 10-21, wherein the region corresponding to positions 345-352 of SEQ ID NO:1 has at least 50%, 60%, 75% or 85% sequence identity with positions 345-352 of SEQ ID NO:1.

[0549] 23. The RSV-F protein according to any one of Embodiments 10-22, which comprises (c) a substitution at position 348 of SEQ ID NO:1 with T or N.

[0550] 24. The RSV-F protein according to any one of Embodiments 10-23, which comprises (c) a substitution at position 348 of SEQ ID NO:1 with N.

[0551] 25. The RSV-F protein according to Embodiment 23 or 24, which comprises a glycan linked to position 348 of SEQ ID NO:1; optionally, wherein the glycan comprises N-acetylglucosamine.

[0552] 26. The RSV-F protein according to any one of Embodiments 10-22, which comprises (d) a substitution at position 348 of SEQ ID NO:1 with N, D, F, H, K, L, N, Q, R, T, W or Y; optionally N, F, H, K, N, Q, R, T, W or Y; optionally N, F, R, W or Y.

[0553] 27. The RSV-F protein according to embodiment 26, which comprises a substitution of N for the amino acid at position 348 of SEQ ID NO:1.

[0554] 28. The RSV-F protein according to any one of embodiments 10-25, which comprises:

[0555] (a) a substitution of T, C, V, I or F; optionally T, C or V; optionally T or V for the amino acid at position 55 of SEQ ID NO:1;

[0556] (b) a substitution of A, P, V, I or F; optionally A or P for the amino acid at position 215 of SEQ ID NO:1; and

[0557] (c) a substitution of T or N for the amino acid at position 348 of SEQ ID NO:1.

[0558] 29. The RSV-F protein according to embodiment 28, which comprises:

[0559] (a) a substitution of T for the amino acid at position 55 of SEQ ID NO:1;

[0560] (b) a substitution of A for the amino acid at position 215 of SEQ ID NO:1; and

[0561] (c) a substitution of N for the amino acid at position 348 of SEQ ID NO:1; wherein the N at position 348 is linked to a glycan; which optionally comprises N-acetylglucosamine.

[0562] 30. The RSV-F protein according to any one of embodiments 10-22, 26 or 27, which comprises:

[0563] (a) a substitution of T, C, V, I or F; optionally T, C or V; optionally T or V for the amino acid at position 55 of SEQ ID NO:1;

[0564] (b) a substitution of A, P, V, I or F; optionally A or P for the amino acid at position 215 of SEQ ID NO:1; and

[0565] (d) a substitution of N, D, F, H, K, L, N, Q, R, T, W or Y; optionally N, F, H, K, N, Q, R, T, W or Y; optionally N, F, R, W or Y for the amino acid at position 348 of SEQ ID NO:1.

[0566] 31. The RSV-F protein according to embodiment 30, which comprises:

[0567] (a) a substitution of T for the amino acid at position 55 of SEQ ID NO:1;

[0568] (b) A substitution at position 215 of SEQ ID NO:1 with A; and

[0569] (d) A substitution at position 348 of SEQ ID NO:1 with N.

[0570] 32. The RSV-F protein according to any one of the foregoing embodiments, which comprises an F2 domain having at least 80%, 85%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with positions 26-108 or 26-109 of SEQ ID NO:1.

[0571] 33. The RSV-F protein according to any one of the foregoing embodiments, which comprises an F1 domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% sequence identity with positions 137-513 of SEQ ID NO:1.

[0572] 34. The RSV-F protein according to any one of the foregoing embodiments, which comprises a heterotrimerization domain at its C-terminus, optionally wherein the heterotrimerization domain is the T4 minor fiber protein foldon domain.

[0573] 35. The RSV-F protein according to embodiments 1-33, which comprises a transmembrane domain at its C-terminus and optionally comprises a cytoplasmic domain at the C-terminus of the transmembrane domain.

[0574] 36. The RSV-F protein according to any one of embodiments 1-33 or 35, which comprises a cytoplasmic domain; wherein, relative to the cytoplasmic domain according to SEQ ID NO:109 or 110, 2-20 residues are deleted from the C-terminus of the cytoplasmic domain of the RSV-F protein.

[0575] 37. The RSV-F protein according to embodiment 35, wherein 3-20 residues are deleted from the C-terminus of the cytoplasmic domain of the RSV-F protein.

[0576] 38. The RSV-F protein according to embodiment 35 or 37, wherein 2-5, such as 2-4, 2-3, 3-4 or 3 residues are deleted from the C-terminus of the cytoplasmic domain of the RSV-F protein.

[0577] 39. The RSV-F protein according to any one of embodiments 35-38, wherein the cytoplasmic domain comprises or consists of the following: (i) the amino acid sequence of positions 10-31 according to SEQ ID NO: 134 or (ii) an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally has the same length as said positions; and wherein the cytoplasmic domain does not contain any residues at the C-terminus of the amino acid sequence of (i) or (ii).

[0578] 40. The RSV-F protein according to any one of embodiments 35-38, wherein the cytoplasmic domain comprises or consists of the following: (i) the amino acid sequence of positions 10-29 according to SEQ ID NO: 135, or (ii) an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally has the same length as said positions; and wherein the cytoplasmic domain does not contain any residues at the C-terminus of the amino acid sequence of (i) or (ii).

[0579] 41. The RSV-F protein according to embodiment 37, wherein 6-13, such as 7-13, 8-12, 9-11, 9-10, 10-11 or 10 residues are deleted from the C-terminus of the cytoplasmic domain of the RSV-F protein.

[0580] 42. The RSV-F protein according to any one of embodiments 35, 37 or 41, wherein the cytoplasmic domain comprises or consists of the following: (i) the amino acid sequence of positions 10-24 according to SEQ ID NO: 136 or (ii) an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to said positions and optionally has the same length as said positions; and wherein the cytoplasmic domain does not contain any residues at the C-terminus of the amino acid sequence of (i) or (ii).

[0581] 43. The RSV-F protein according to embodiment 37, wherein 14-16, such as 14-15 or 15-16 or 15 residues are deleted from the C-terminus of the cytoplasmic domain of the RSV-F protein.

[0582] 44. The RSV-F protein according to any one of embodiments 35, 37 or 43, wherein the cytoplasmic domain comprises or consists of the following: (i) the amino acid sequence of positions 10-19 of SEQ ID NO: 137, or (ii) an amino acid sequence that is at least 60%, 70%, 80% or 90% identical to said positions and optionally of the same length as said positions; and wherein the cytoplasmic domain does not contain any residues at the C-terminus of the amino acid sequence of (i) or (ii).

[0583] 45. The RSV-F protein according to embodiment 37, wherein 16-20, such as 17-20, 18-20 or 19-20 residues are deleted from the C-terminus of the cytoplasmic domain of the RSV-F protein.

[0584] 46. The RSV-F protein according to embodiment 45, wherein 20 residues are deleted from the C-terminus of the cytoplasmic tail of the RSV-F domain.

[0585] 47. The RSV-F protein according to any one of embodiments 35, 37, 45 or 46, wherein the cytoplasmic tail comprises or consists of the following: (i) the amino acid sequence of positions 10-14 of SEQ ID NO: 138, or (ii) an amino acid sequence that is at least 60% or 80% identical to said positions and optionally of the same length as said positions; and wherein the cytoplasmic tail does not contain any residues at the C-terminus of the amino acid sequence of (i) or (ii).

[0586] 48. The RSV-F protein according to any one of embodiments 35-47, wherein optionally in human fibroblasts, optionally in human foreskin fibroblasts, optionally in primary human BJ cells, optionally the ATCC CRL-2522 cell line, relative to the expression of the RSV-F protein having the same amino acids but without such deletions (such as the RSV-F protein comprising the cytoplasmic domain according to SEQ ID NO: 109 or 110) in the pre-fusion trimeric form, the deletion increases the cell surface expression of the RSV-F protein in this form from the nucleic acid.

[0587] 49. The RSV-F protein according to embodiment 48, wherein the increased cell surface expression persists for a period of at least 24, 48, 72 or 96 hours.

[0588] 50. The RSV-F protein according to any one of the preceding embodiments, wherein the signal peptide is absent from the RSV-F protein, optionally as a result of signal peptide cleavage, optionally wherein the signal peptide is or corresponds to positions 1-25 of SEQ ID NO: 1.

[0589] 51. The RSV-F protein according to any of the preceding embodiments, wherein the p27 peptide is absent from the RSV-F protein, optionally as a result of furin processing, optionally wherein the p27 peptide is or corresponds to positions 110-136 of SEQ ID NO:1.

[0590] 52. The RSV-F protein according to any of the preceding embodiments, wherein the RSV-F protein comprises an E residue at position 66 and a P residue at position 101 of SEQ ID NO:1.

[0591] 53. The RSV-F protein according to any of the preceding embodiments, which further comprises, relative to SEQ ID NO:1:

[0592] a substitution at position 152 with R, L or W; optionally R or W; optionally a substitution with R;

[0593] a substitution at position 210 with H, A, F, K, N, W or Y; optionally H, F, K, N, W or Y; optionally H, F or Y; optionally a substitution with H;

[0594] optionally, a substitution at position 211 with N;

[0595] a substitution at position 241 with N

[0596] a substitution at position 315 with I or V; optionally a substitution with I;

[0597] a substitution at position 346 with Q, D, H, K, N, R, S or W; optionally Q, D, H, K, N, R or S; optionally a substitution with Q;

[0598] a substitution at position 419 with D, N, S or T; optionally D or T; optionally a substitution with D;

[0599] optionally, a substitution at position 445 with D;

[0600] a substitution at position 455 with V or I; optionally a substitution with V;

[0601] and / or, optionally and,

[0602] a substitution at position 459 with M.

[0603] 54. The RSV-F protein according to any of the preceding embodiments, which further comprises, relative to SEQ ID NO:1:

[0604] a substitution at position 152 with R, L or W; optionally R or W; optionally a substitution with R;

[0605] optionally, a substitution at position 211 with N;

[0606] A substitution with I or V at position 315; optionally a substitution with I;

[0607] A substitution with Q, D, H, K, N, R, S or W at position 346; optionally a substitution with Q, D, H, K, N, R or S; optionally a substitution with Q;

[0608] Optionally, a substitution with D at position 445;

[0609] A substitution with V or I at position 455; optionally a substitution with V;

[0610] And / or, optionally and;

[0611] A substitution with M at position 459.

[0612] 55. The RSV-F protein according to any one of the foregoing embodiments, which further comprises, relative to SEQ ID NO:1:

[0613] A substitution with I or V at position 315; optionally a substitution with I;

[0614] A substitution with N at position 241

[0615] A substitution with V or I at position 455; optionally a substitution with V;

[0616] And / or, optionally and;

[0617] A substitution with M at position 459.

[0618] 56. The RSV-F protein according to any one of the foregoing embodiments, which further comprises, relative to SEQ ID NO:1:

[0619] A substitution with I or V at position 315; optionally a substitution with I;

[0620] A substitution with V or I at position 455; optionally a substitution with V;

[0621] And / or, optionally and;

[0622] A substitution with M at position 459.

[0623] 57. The RSV-F protein according to any one of the foregoing embodiments, which comprises a substitution with R at position 152 of SEQ ID NO:1, and / or a substitution with Q at position 346 of SEQ ID NO:1.

[0624] 58. The RSV-F protein according to any of the preceding embodiments, which comprises a substitution of N at position 211 of SEQ ID NO:1, and / or a substitution of D at position 445 of SEQ ID NO:1, and optionally comprises both substitutions.

[0625] 59. The RSV-F protein according to any of the preceding embodiments, wherein the RSV-F protein is subtype A.

[0626] 60. The RSV-F protein according to any one of embodiments 1-58, wherein the RSV-F protein is subtype B.

[0627] 61. The RSV-F protein according to any of the preceding embodiments, which is specifically bound by a pre-fusion mAb, as measured by SPR, with a K D less than 10 nM; optionally 1 pM - 10 nM.

[0628] 62. The RSV-F protein according to any of the preceding embodiments, which is specifically bound by a pre-fusion mAb comprising LC and HC according to SEQ ID NOs: 2 and 3 respectively, as measured by SPR, with a K D less than 1000, 900, 800, 700, 650, 600, 550, 100, 90, 80, 70, 60, 50 or 35 pM; wherein the RSV-F protein is in trimeric form.

[0629] 63. The RSV-F protein according to any of the preceding embodiments, which is specifically bound by a pre-fusion mAb comprising LC and HC according to SEQ ID NOs: 4 and 5 respectively, as measured by SPR, with a K D less than 200, 180, 160, 140, 130, 100, 95, 90, 85, 80 or 70 pM.

[0630] 64. The RSV-F protein according to any of the preceding embodiments, which is specifically bound by a pre-fusion mAb comprising LC and HC according to SEQ ID NOs: 8 and 9 respectively, as measured by SPR, with a K D less than 150, 120, 110, 100, 105, 95, 90, 80, 75, 70, 60, 55, 50 or 45 pM.

[0631] 65. The RSV-F protein according to any of the preceding embodiments, which is specifically bound by a pre-fusion mAb comprising LC and HC according to SEQ ID NOs: 8 and 9 respectively, as measured by SPR, with a K DLess than 150, 120, 110, 100, 105, 95, 90, 80, 75, 70, 60, 55, 50 or 45 pM.

[0632] 66. The RSV-F protein according to any one of the foregoing embodiments, which is specifically bound by an mAb comprising LC and HC according to SEQ ID NOs: 6 and 7 respectively, and as measured by SPR, its K D Less than 200, 180, 160, 140, 120, 110, 100, 95, 80, 70, 60, 55, 50, 45 or 40 pM.

[0633] 67. A recombinant RSV-F protein in a pre-fusion conformation, which comprises at least one mutation relative to the wild-type RSV-F according to SEQ ID NO: 1, wherein at least one mutation does not introduce an artificial disulfide bond or P residue into the wild-type protein.

[0634] 68. The RSV-F protein according to embodiment 67, which comprises the features of any one of embodiments 1-66, provided that a P residue is not introduced into the protein by the at least one mutation.

[0635] 69. A trimer which comprises three RSV-F proteins according to any one of the foregoing embodiments.

[0636] 70. A nucleic acid which encodes an RSV-F protein according to any one of embodiments 1-68.

[0637] 71. The nucleic acid according to embodiment 70, wherein the nucleic acid is a viral vector, or is comprised within a viral vector; optionally wherein the viral vector is an adenovirus vector.

[0638] 72. The nucleic acid according to embodiment 70, wherein the nucleic acid is DNA; optionally wherein the DNA is a DNA plasmid.

[0639] 73. The nucleic acid according to embodiment 70, wherein the nucleic acid is RNA.

[0640] 74. The RNA according to embodiment 73, which is a non-self-replicating RNA.

[0641] 75. The RNA according to embodiment 73, which is a self-replicating RNA.

[0642] 76. The RNA according to any one of embodiments 73-75, which comprises, in the 5' to 3' direction: a 5' cap, a 5' UTR, an open reading frame encoding at least one RSV-F protein according to any one of embodiments 1-68, a 3' UTR, and a 3' polyA tail.

[0643] 77. The RNA according to embodiment 76, wherein the 5' cap comprises 7'-methylguanosine linked 5' to 5' to the 5' first ribonucleoside via a triphosphate bridge, and wherein the first 5' ribonucleoside comprises 2'-O-methylated ribose (2'-O-Me).

[0644] 78. The RNA according to embodiment 76 or 77, wherein the 3' polyA tail comprises a continuous stretch of 100 - 500 A ribonucleotides.

[0645] 79. The RNA according to embodiment 76 or 77, wherein the 3' polyA tail comprises at least two non - continuous stretches of A ribonucleotides; optionally having lengths of 25 - 35 and 65 - 90 ribonucleotides respectively; optionally oriented in the 5' to 3' direction.

[0646] 80. The RNA according to any one of embodiments 73 - 79, which comprises modified ribonucleotides.

[0647] 81. The RNA according to embodiment 80, wherein the modified ribonucleotide is 1mΨ.

[0648] 82. The RNA according to embodiment 81, wherein the RNA comprises 1mΨ and does not comprise standard U ribonucleotides or other modified U ribonucleotides; optionally wherein the RNA comprises 1mΨ and does not comprise standard U ribonucleotides or other modified ribonucleotides.

[0649] 83. The RNA according to any one of embodiments 73 - 82, which has a GC content of 55 - 70%.

[0650] 84. The RNA according to any one of embodiments 73 - 82, which has a GC content of 40 - 60%.

[0651] 85. A vector, which comprises a nucleic acid according to any one of embodiments 70, 72 or 73 - 83.

[0652] 86. The vector according to embodiment 85, which is a lipid nanoparticle.

[0653] 87. The lipid nanoparticle according to embodiment 86, which comprises a mixture of a cationic lipid, a neutral lipid, a sterol and a polymer - conjugated lipid.

[0654] 88. The lipid nanoparticle according to embodiment 87, wherein the pKa of the cationic lipid is 5.0 - 8.0; optionally 5.0 - 7.6.

[0655] 89. The lipid nanoparticle according to embodiment 87 or 88, wherein the cationic lipid comprises a tertiary amine group.

[0656] 90. The lipid nanoparticle according to any one of embodiments 87 - 89, wherein the polymer-conjugated lipid is a PEGylated lipid; optionally wherein the average molecular weight of the PEG is 1 - 3 kDa.

[0657] 91. The lipid nanoparticle according to embodiment 90, wherein the weight-average molecular weight of the PEG is 1 - 3 kDa.

[0658] 92. The lipid nanoparticle according to any one of embodiments 87 - 90, wherein the sterol is cholesterol or a cholesterol-based lipid.

[0659] 93. The lipid nanoparticle according to any one of embodiments 87 - 92, which comprises (in mole %) 30 - 60% cationic lipid, 35 - 70% sterol, 0.8 - 4.0% polymer-conjugated lipid, and 0 - 15% neutral lipid; optionally 40 - 50% cationic lipid, 41 - 49% sterol, 1.0 - 3.0% polymer-conjugated lipid, and 8.0 - 11.0% neutral lipid.

[0660] 94. A pharmaceutical composition, which comprises the RSV-F protein according to any one of embodiments 1 - 68, the trimer according to embodiment 69, the nucleic acid according to any one of embodiments 70 - 83, or the carrier according to any one of embodiments 85 - 93; optionally comprises a pharmaceutically acceptable excipient; optionally further comprises an adjuvant.

[0661] 95. A vaccine composition, which comprises the RSV-F protein according to any one of embodiments 1 - 68, the...

Claims

1. A respiratory syncytial virus fusion (RSV-F) protein in a pre-fusion conformation, which is mutated relative to SEQ ID NO:1; wherein the RSV-F protein comprises at least one mutation relative to SEQ ID NO:1 in the region corresponding to positions 217-239 of SEQ ID NO:1; wherein the at least one mutation is introduced by substitution or insertion of a residue containing a hydrogen bond donor and / or acceptor moiety in the side chain.

2. The RSV-F protein according to claim 1, wherein the region corresponding to positions 217-239 of SEQ ID NO:1 comprises an α-helix.

3. The RSV-F protein according to claim 1 or 2, wherein the region corresponding to positions 217-239 of SEQ ID NO:1 has at least 80%, 85%, 90% or 95% sequence identity with positions 217-239 of SEQ ID NO:

1.

4. The RSV-F protein according to any one of the preceding claims, wherein the at least one mutation introduces, by substitution or insertion, a residue selected from K, R, W, N, Q, H, S, T and Y; optionally K, R, Q and N; optionally K and R; optionally K into the region corresponding to positions 220-235 of SEQ ID NO:1; optionally positions 227-232; optionally positions 228-232.

5. The RSV-F protein according to claim 4, which comprises a substitution at position 228 of SEQ ID NO:1 with K, R, Q or N; optionally K, R or Q; optionally K or R; optionally K; and / or a substitution at position 232 of SEQ ID NO:1 with N.

6. The RSV-F protein according to claim 5, which comprises a substitution at position 228 (N) of SEQ ID NO:1 with K, R, Q or N; optionally K, R or Q; optionally K or R.

7. The RSV-F protein according to claim 6, which comprises a substitution at position 228 (N) of SEQ ID NO:1 with K.

8. The RSV-F protein according to any one of the preceding claims, which comprises: (a) a substitution at position 55 of SEQ ID NO:1 with T, C, V, I or F; optionally T, C or V; optionally T or V; (b) a substitution at position 215 of SEQ ID NO:1 with A, P, V, I or F; optionally A or P; and / or, optionally and, (c) a substitution at position 348 of SEQ ID NO: with T or N.

9. The RSV-F protein according to claim 8, which comprises: (a) a substitution at position 55 of SEQ ID NO:1 with T; (b) a substitution at position 215 of SEQ ID NO:1 with A; and / or, optionally and, (c) a substitution at position 348 of SEQ ID NO:1 with N.

10. The RSV-F protein according to claim 8 or 9, which comprises a glycan linked to position 348; optionally comprising N-acetylglucosamine.

11. The RSV-F protein according to any one of the preceding claims, which comprises an F2 domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with positions 26-108 or 26-109 of SEQ ID NO:

1.

12. The RSV-F protein according to any one of the preceding claims, which comprises an F1 domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2% or 99.5% sequence identity with positions 137-513 of SEQ ID NO:

1.

13. The RSV-F protein according to any one of the preceding claims, which comprises a heterotrimerization domain at its C-terminus and / or at the C-terminus of the F1 domain; optionally wherein the heterotrimerization domain is the T4 minor fiber protein foldon domain.

14. The RSV-F protein according to any one of claims 1-12, which comprises a transmembrane domain at its C-terminus and / or at the C-terminus of the F1 domain; and optionally a cytoplasmic domain at the C-terminus of the transmembrane domain.

15. The RSV-F protein according to any one of claims 1-12 or 14, wherein the RSV-F protein comprises a cytoplasmic domain; wherein 2-20 residues are deleted from the C-terminus of the cytoplasmic domain of the RSV-F protein relative to the cytoplasmic domain according to SEQ ID NO:109 or 110.

16. The RSV-F protein according to claim 15, wherein 16-20, 17-20, 18-20, 19-20 or 20 residues are deleted from the C-terminus of the cytoplasmic domain of the RSV-F protein.

17. The RSV-F protein according to any one of the preceding claims, wherein the signal peptide is absent from the RSV-F protein, optionally as a result of signal peptide cleavage, optionally wherein the signal peptide is or corresponds to positions 1-25 of SEQ ID NO:

1.

18. The RSV-F protein according to any one of the preceding claims, wherein the p27 peptide is absent from the RSV-F protein, optionally as a result of furin processing, optionally wherein the p27 peptide is or corresponds to positions 110-136 of SEQ ID NO:

1.

19. The RSV-F protein according to any one of the preceding claims, wherein the RSV-F protein comprises an E residue at position 66 and a P residue at position 101 of SEQ ID NO:

1.

20. The RSV-F protein according to any one of the preceding claims, wherein the RSV-F protein is of subtype A.

21. The RSV-F protein according to any one of claims 1-19, wherein the RSV-F protein is of subtype B.

22. A homotrimer comprising three RSV-F proteins according to any one of the preceding claims, which have the same amino acid sequence.

23. A nucleic acid encoding the RSV-F protein according to any one of claims 1-21.

24. The nucleic acid according to claim 23, wherein the nucleic acid is RNA.

25. A lipid nanoparticle comprising the nucleic acid according to claim 23 or 24.

26. A pharmaceutical composition comprising the RSV-F protein according to any one of claims 1-21, the trimer according to claim 22, the nucleic acid according to claim 23 or 24, or the lipid nanoparticle according to claim 25; optionally for medical use.

27. The pharmaceutical composition for the use according to claim 26, for a method of vaccinating a subject against RSV; optionally wherein the subject is: a human infant, optionally 2-6 months old; an elderly human, optionally ≥60 years old; or a pregnant human female, optionally ≥28 weeks pregnant.

28. A method of inducing an immune response against RSV in a subject, comprising administering to the subject an immunologically effective amount of the RSV-F protein according to any one of claims 1-21, the trimer according to claim 22, the nucleic acid according to claim 23 or 24, the lipid nanoparticle according to claim 25, or the pharmaceutical composition according to claim 26.

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